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Sample records for cell-free fetal dna

  1. Cell-Free Fetal DNA and Cell-Free Total DNA Levels in Spontaneous Abortion with Fetal Chromosomal Aneuploidy

    OpenAIRE

    Ji Hyae Lim; Min Hyoung Kim; You Jung Han; Da Eun Lee; So Yeon Park; Jung Yeol Han; Moon Young Kim; Hyun Mee Ryu

    2013-01-01

    BACKGROUND: Cell-free fetal DNA and cell-free total DNA in maternal circulation have been proposed as potential markers for noninvasive monitoring of the placental condition during the pregnancy. However, the correlation of and change in cell-free fetal DNA and cell-free total DNA in spontaneous abortion (SA) with fetal chromosomal aneuploidy have not yet been reported. Therefore, we investigated cell-free fetal DNA and cell-free total DNA levels in SA women with fetal chromosomal aneuploidy....

  2. Cell-free fetal DNA and cell-free total DNA levels in spontaneous abortion with fetal chromosomal aneuploidy.

    Directory of Open Access Journals (Sweden)

    Ji Hyae Lim

    Full Text Available BACKGROUND: Cell-free fetal DNA and cell-free total DNA in maternal circulation have been proposed as potential markers for noninvasive monitoring of the placental condition during the pregnancy. However, the correlation of and change in cell-free fetal DNA and cell-free total DNA in spontaneous abortion (SA with fetal chromosomal aneuploidy have not yet been reported. Therefore, we investigated cell-free fetal DNA and cell-free total DNA levels in SA women with fetal chromosomal aneuploidy. METHODOLOGY/PRINCIPAL FINDINGS: A nested case-control study was conducted with maternal plasma collected from 268 women in their first trimester of pregnancy. Subjects included 41 SA with normal fetal karyotype, 26 SA with fetal chromosomal aneuploidy, and 201 normal controls. The unmethylated PDE9A gene was used to measure the maternal plasma levels of cell-free fetal DNA. The GAPDH gene was used to measure the maternal plasma levels of cell-free total DNA. The diagnostic accuracy was measured using receiver-operating characteristic (ROC curves. Levels of cell-free fetal DNA and cell-free total DNA were significantly higher in both SA women with normal fetal karyotype and SA women with fetal chromosomal aneuploidy in comparison with the normal controls (P<0.001 in both. The correlation between cell-free fetal DNA and cell-free total DNA levels was stronger in the normal controls (r = 0.843, P<0.001 than in SA women with normal karyotype (r = 0.465, P = 0.002 and SA women with fetal chromosomal aneuploidy (r = 0.412, P = 0.037. The area under the ROC curve for cell-free fetal DNA and cell-free total DNA was 0.898 (95% CI, 0.852-0.945 and 0.939 (95% CI, 0.903-0.975, respectively. CONCLUSIONS: Significantly high levels of cell-free fetal DNA and cell-free total DNA were found in SA women with fetal chromosomal aneuploidy. Our findings suggest that cell-free fetal DNA and cell-free total DNA may be useful biomarkers for the prediction of SA

  3. Noninvasive Fetal Sex Determination Using Cell-Free Fetal DNA

    Science.gov (United States)

    Devaney, Stephanie A.; Palomaki, Glenn E.; Scott, Joan A.; Bianchi, Diana W.

    2015-01-01

    Context Noninvasive prenatal determination of fetal sex using cell-free fetal DNA provides an alternative to invasive techniques for some heritable disorders. In some countries this testing has transitioned to clinical care, despite the absence of a formal assessment of performance. Objective To document overall test performance of noninvasive fetal sex determination using cell-free fetal DNA and to identify variables that affect performance. Data Sources Systematic review and meta-analysis with search of PubMed (January 1, 1997–April 17, 2011) to identify English-language human studies reporting primary data. References from review articles were also searched. Study Selection and Data Extraction Abstracts were read independently to identify studies reporting primary data suitable for analysis. Covariates included publication year, sample type, DNA amplification methodology, Y chromosome sequence, and gestational age. Data were independently extracted by 2 reviewers. Results From 57 selected studies, 80 data sets (representing 3524 male-bearing pregnancies and 3017 female-bearing pregnancies) were analyzed. Overall performance of the test to detect Y chromosome sequences had the following characteristics: sensitivity, 95.4% (95% confidence interval [CI], 94.7%–96.1%) and specificity, 98.6% (95% CI, 98.1%–99.0%); diagnostic odds ratio (OR), 885; positive predictive value, 98.8%; negative predictive value, 94.8%; area under curve (AUC), 0.993 (95% CI, 0.989–0.995), with significant interstudy heterogeneity. DNA methodology and gestational age had the largest effects on test performance. Methodology test characteristics were AUC, 0.988 (95% CI, 0.979–0.993) for polymerase chain reaction (PCR) and AUC, 0.996 (95% CI, 0.993–0.998) for real-time quantitative PCR (RTQ-PCR) (P=.02). Gestational age test characteristics were AUC, 0.989 (95% CI, 0.965–0.998) (20 weeks) (P=.02 for comparison of diagnostic ORs across age ranges). RTQ-PCR (sensitivity, 96

  4. Implementing Prenatal Diagnosis Based on Cell-Free Fetal DNA: Accurate Identification of Factors Affecting Fetal DNA Yield

    OpenAIRE

    Barrett, A. N.; Zimmermann, B. G.; Wang, D.; Holloway, A.; Chitty, L S

    2011-01-01

    Objective: Cell-free fetal DNA is a source of fetal genetic material that can be used for non-invasive prenatal diagnosis. Usually constituting less than 10% of the total cell free DNA in maternal plasma, the majority is maternal in origin. Optimizing conditions for maximizing yield of cell-free fetal DNA will be crucial for effective implementation of testing. We explore factors influencing yield of fetal DNA from maternal blood samples, including assessment of collection tubes containing ce...

  5. Implementing prenatal diagnosis based on cell-free fetal DNA: accurate identification of factors affecting fetal DNA yield.

    Directory of Open Access Journals (Sweden)

    Angela N Barrett

    Full Text Available OBJECTIVE: Cell-free fetal DNA is a source of fetal genetic material that can be used for non-invasive prenatal diagnosis. Usually constituting less than 10% of the total cell free DNA in maternal plasma, the majority is maternal in origin. Optimizing conditions for maximizing yield of cell-free fetal DNA will be crucial for effective implementation of testing. We explore factors influencing yield of fetal DNA from maternal blood samples, including assessment of collection tubes containing cell-stabilizing agents, storage temperature, interval to sample processing and DNA extraction method used. METHODS: Microfluidic digital PCR was performed to precisely quantify male (fetal DNA, total DNA and long DNA fragments (indicative of maternal cellular DNA. Real-time qPCR was used to assay for the presence of male SRY signal in samples. RESULTS: Total cell-free DNA quantity increased significantly with time in samples stored in K(3EDTA tubes, but only minimally in cell stabilizing tubes. This increase was solely due to the presence of additional long fragment DNA, with no change in quantity of fetal or short DNA, resulting in a significant decrease in proportion of cell-free fetal DNA over time. Storage at 4 °C did not prevent these changes. CONCLUSION: When samples can be processed within eight hours of blood draw, K(3EDTA tubes can be used. Prolonged transfer times in K(3EDTA tubes should be avoided as the proportion of fetal DNA present decreases significantly; in these situations the use of cell stabilising tubes is preferable. The DNA extraction kit used may influence success rate of diagnostic tests.

  6. Modifying Risk of Aneuploidy with a Positive Cell-Free Fetal DNA Result.

    Science.gov (United States)

    Long, A Ashleigh; Abuhamad, Alfred Z; Warsof, Steven L

    2016-06-01

    Noninvasive genomic assessments of the fetus while in utero have been made possible by the analysis of cell-free fetal DNA fragments from the serum of pregnant women, as part of a noninvasive prenatal testing screening strategy. Between 7% and 10% of total cell-free DNA in the maternal blood comes from placental trophoblasts, allowing for identification of the DNA associated with the fetal component of the placenta. Using simple venipuncture in the outpatient setting, this cell-free, extracellular fetal DNA can be isolated in the maternal serum from a single blood draw as early as the seventh week of gestation. PMID:27235910

  7. Cell free fetal DNA testing in maternal blood of Romanian pregnant women

    OpenAIRE

    Radoi, Viorica E; Camil L Bohiltea; Roxana E Bohiltea; Dragos N Albu

    2015-01-01

    Background: The discovery of circulating fetal DNA in maternal blood led to the discovery of new strategies to perform noninvasive testing for prenatal diagnosis. Objective: The purpose of the study was to detect fetal aneuploidy at chromosomes 13, 18, 21, X, and Y by analysis of fetal cell-free DNA from maternal blood, without endangering pregnancy. Materials and Methods: This retrospective study has been performed in Bucharest at Medlife Maternal and Fetal Medicine Department between ...

  8. Understanding the Limitations of Circulating Cell Free Fetal DNA: An Example of Two Unique Cases.

    Science.gov (United States)

    Clark-Ganheart, Cecily A; Iqbal, Sara N; Brown, Donna L; Black, Susan; Fries, Melissa H

    2014-05-01

    Circulating cell free fetal DNA (cffDNA) is an effective screening modality for fetal aneuploidy. We report two cases of false positive results. The first case involves a female, with self-reported Down syndrome. CffDNA returned positive for trisomy 18 leading to a maternal diagnosis of mosaicism chromosome 18 with normal fetal karyotype. The second case involves a patient with an anomalous fetal ultrasound and cffDNA positive for trisomy 13. Amniocentesis demonstrated a chromosome 8p duplication/deletion. False positive cffDNA may arise in clinical scenarios where diagnostic testing is clearly indicated. Practitioners should recognize the limitations of cffDNA. PMID:25298847

  9. Non-invasive prenatal diagnosis of fetal trisomy 21 using cell-free fetal DNA in maternal blood

    OpenAIRE

    Lim, Ji Hyae; Park, So Yeon; Ryu, Hyun Mee

    2013-01-01

    Since the existence of cell-free fetal DNA (cff-DNA) in maternal circulation was discovered, it has been identified as a promising source of fetal genetic material in the development of reliable methods for non-invasive prenatal diagnosis (NIPD) of fetal trisomy 21 (T21). Currently, a prenatal diagnosis of fetal T21 is achieved through invasive techniques, such as chorionic villus sampling or amniocentesis. However, such invasive diagnostic tests are expensive, require expert technicians, and...

  10. Evaluation of prenatal RHD typing strategies on cell-free fetal DNA from maternal plasma

    NARCIS (Netherlands)

    M.G.H.M. Grootkerk-Tax; A.A. Soussan; M. de Haas; P.A. Maaskant-van Wijk; C.E. van der Schoot

    2006-01-01

    BACKGROUND: The discovery of cell-free fetal DNA in maternal plasma led to the development of assays to predict the fetal D status with RHD-specific sequences. Few assays are designed in such a way that the fetus can be typed in RHD psi mothers and that RHD psi fetuses are correctly typed. Owing to

  11. Cell-free fetal DNA in amniotic fluid supernatant for prenatal diagnosis.

    Science.gov (United States)

    Soltani, M; Nemati, M; Maralani, M; Estiar, M A; Andalib, S; Fardiazar, Z; Sakhinia, E

    2016-01-01

    In widespread conviction, amniotic fluid is utilized for prenatal diagnosis. Amniotic fluid supernatant is usually discarded, notwithstanding being a good source of fetal DNA. The aim of the present study was to assess cell-free fetal DNA extracted from amniotic fluid supernatant for application in prenatal diagnosis such as gender determination and early diagnosis of β-thalassemia. Samples of amniotic fluid of 70 pregnant women were collected and went through routine tests along with tests for cell-free fetal DNA from amniotic fluid supernatant. The DNA in the amniotic fluid supernatant was extracted and analyzed for gender determination by PCR and Real-time PCR. ARMS-PCR was applied to test early diagnosis of IVS II-I mutation (common β-thalassemia mutation) and E7V mutation for sickle cell anemia using DNA extracted from the amniotic fluid supernatant. Using the cell-free fetal DNA extracted from the amniotic fluid supernatant, the sensitivity of PCR and Real-time PCR for gender detection was compared with the routine cytogenetic method. The fetus tested for sickle cell anemia and β-thalassemia was observed to be healthy but heterozygous for IVS II-I mutation. The findings indicated that cell-free fetal DNA from amniotic fluid supernatant can be a good source of fetal DNA and be used in early prenatal diagnosis since because of its fast and accurate application. Therefore, it would be suggested that the amniotic fluid supernatant's disposal is prevented because if the tests needs to be repeated, cell-free fetal DNA extracted from the amniotic fluid supernatant can be used as an alternative source for prenatal diagnosis. PMID:27188728

  12. Relationship of circulating cell-free DNA levels to cell-free fetal DNA levels, clinical characteristics and laboratory parameters in preeclampsia

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    Mézes Miklós

    2009-01-01

    Full Text Available Abstract Background The aim of our study was to examine whether increased circulating total cell-free DNA levels are related to the clinical characteristics and standard laboratory parameters of preeclamptic patients, to markers of inflammation, endothelial activation or injury, oxidative stress and to cell-free fetal DNA levels. Methods Circulating total cell-free DNA was measured by real-time quantitative PCR in plasma samples obtained from 67 preeclamptic and 70 normotensive pregnant women. Standard laboratory parameters, C-reactive protein, plasma von Willebrand factor antigen, plasma fibronectin, plasma malondialdehyde and cell-free fetal DNA levels were also determined. Results and Conclusion Circulating total cell-free and fetal deoxyribonucleic acid levels were significantly elevated in pregnancies complicated by preeclampsia (median: 11.395 vs. 32.460 and 0.001 vs. 0.086 pg/μl; P < .001. The quantity of plasma total cell-free DNA did not correlate with most of the laboratory parameters, except for serum aspartate aminotransferase and alanine aminotransferase activities (correlation coefficient: 0.31; P = 0.012 and 0.46; P < .001. There was no correlation with clinical characteristics, including body mass index. The releases of both free fetal and total cell-free deoxyribonucleic acid were found to be affected in preeclampsia. Hepatocellular necrosis seems to be responsible - at least partly - for increased circulating total DNA levels in preeclampsia, as suggested by the significant correlation with liver enzyme activities.

  13. Both maternal and fetal cell-free DNA in plasma fluctuate.

    Science.gov (United States)

    Hahn, S; Zhong, X Y; Bürk, M R; Troeger, C; Kang, A; Holzgreve, W

    2001-09-01

    Elevations in the concentration of cell-free fetal DNA in maternal plasma have recently been determined in various pregnancy-related disorders, including preeclampsia, preterm labor, and polyhydramnios. In addition, almost 2-fold increments in cell-free fetal DNA levels have been recorded in pregnancies with certain aneuploid fetuses, in particular trisomy 21. These findings have led to the speculation that quantitative assessment of circulatory fetal DNA may be useful in the noninvasive prenatal diagnosis of certain fetal genetic constellations or pregnancy-related disorders. A premise for any quantitative analysis is that the quantity of the analyte being assayed does not vary greatly over time. As this aspect has not been examined for circulatory DNA levels, we examined these in normal healthy individuals as well as in pregnant women. Our data indicate that severalfold alterations in circulatory DNA amounts do occur over short periods of time. Of particular note is that we observed almost 2-fold variations in free fetal DNA levels over a period of 3 days, which are in a similar range to the elevations noted in aneuploid pregnancies. Our results, therefore, imply that caution should be used when using small increments in circulatory fetal DNA concentrations for potential diagnostic applications. PMID:11708468

  14. Integrating stakeholder perspectives into the translation of cell-free fetal DNA testing for aneuploidy

    OpenAIRE

    Sayres, Lauren C.; Allyse, Megan; Cho, Mildred K.

    2012-01-01

    Background The translation of novel genomic technologies from bench to bedside enjoins the comprehensive consideration of the perspectives of all stakeholders who stand to influence, or be influenced by, the translational course. Non-invasive prenatal aneuploidy testing that utilizes cell-free fetal DNA (cffDNA) circulating in maternal blood is one example of an innovative technology that promises significant benefits for its intended end users; however, it is currently uncertain whether it w...

  15. The cell-free fetal DNA fraction in maternal blood decreases after physical activity

    DEFF Research Database (Denmark)

    Schlütter, Jacob Mørup; Hatt, Lotte; Bach, Cathrine; Kirkegaard, Ida; Kølvraa, Steen; Uldbjerg, Niels

    2014-01-01

    cycling with a pulse-rate of 150 beats per minute. The concentrations of cffDNA (DYS14) and cfDNA (RASSF1A) were assessed using quantitative real-time polymerase chain reaction. RESULTS: The fetal fraction decreased significantly in all participants after physical activity (p < 0.01), a decrease varying......OBJECTIVE: If noninvasive prenatal testing using next generation sequencing is to be effective for pregnant women, a cell-free fetal DNA (cffDNA) fraction above 4% is essential unless the depth of sequencing is increased. This study's objective is to determine whether physical activity has an...... effect on the proportion of cell-free DNA (cfDNA) arising from the fetus (fetal fraction). METHODS: Nine pregnant women carrying male fetuses at gestational age 12(+0)  weeks to 14(+6)  weeks were included. Plasma from nine pregnant women was drawn prior to, immediately after, and 30 min after 30 min of...

  16. Cell free fetal DNA testing in maternal blood of Romanian pregnant women

    Directory of Open Access Journals (Sweden)

    Viorica E Radoi

    2015-10-01

    Full Text Available Background: The discovery of circulating fetal DNA in maternal blood led to the discovery of new strategies to perform noninvasive testing for prenatal diagnosis. Objective: The purpose of the study was to detect fetal aneuploidy at chromosomes 13, 18, 21, X, and Y by analysis of fetal cell-free DNA from maternal blood, without endangering pregnancy. Materials and Methods: This retrospective study has been performed in Bucharest at Medlife Maternal and Fetal Medicine Department between 2013-2014. In total 201 women were offered noninvasive prenatal test. Maternal plasma samples were collected from women at greater than 9 weeks of gestation after informed consent and genetics counseling. Results: From 201 patients; 28 (13.93% had screening test with high risk for trisomy 21, 116 (57.71% had advanced maternal age, 1 (0.49% had second trimester ultrasound markers and the remaining 56 patients (27.86% performed the test on request. Of those patients, 189 (94.02% had a “low risk” result (99% risk all for trisomy 21 (T21. T21 was confirmed by amniocentesis in 1 patient and the other 4 patients declined confirmation. The 7 remaining patients (3.48% had a low fetal fraction of DNA. Conclusion: It is probably that prenatal diagnosis using fetal DNA in maternal blood would play an increasingly role in the future practice of prenatal testing because of high accuracy.

  17. Evaluation of a Modified DNA Extraction Method for Isolation of Cell-Free Fetal DNA from Maternal Serum

    OpenAIRE

    Keshavarz, Zeinab; Moezzi, Leili; Ranjbaran, Reza; Aboualizadeh, Farzaneh; Behzad-Behbahani, Abbas; Abdullahi, Masooma; Sharifzadeh, Sedigheh

    2015-01-01

    Background: Discovery of short cell free fetal DNA (cffDNA) fragments in maternal plasma has created major changes in the field of prenatal diagnosis. The use of cffDNA to set up noninvasive prenatal test is limited due to the low concentration of fetal DNA in maternal plasma therefore, employing a high efficiency extraction method leads to more accurate results. The aim of this study was to evaluate the efficiency of Triton/Heat/Phenol (THP) protocol in comparison with the QIAamp DNA Blood m...

  18. Cell-Free Fetal DNA, Telomeres, and the Spontaneous Onset of Parturition.

    Science.gov (United States)

    Phillippe, Mark

    2015-10-01

    Multiple previous reports have provided compelling support for the premise that spontaneous parturition is mediated by activation of inflammation-related signaling pathways leading to increased secretion of cytokines and chemokines, the influx of neutrophils and macrophages into the pregnant uterus, increased production of uterine activation proteins (eg, connexin-43, cyclo-oxygenase-2, oxytocin receptors, etc), activation of matrix metalloproteinases, and the release of uterotonins leading to cervical ripening, membrane rupture, and myometrial contractions. The missing link has been the fetal/placental signal that triggers these proinflammatory events in the absence of microbial invasion and intrauterine infection. This article reviews the biomedical literature regarding the increase in cell-free fetal DNA (cffDNA), which is released during apoptosis in the placenta and fetal membranes at term, the ability of apoptosis modified vertebrate DNA to stimulate toll-like receptor-9 (TLR9) leading to increased release of cytokines and chemokines, and the potential "fail-safe" role for the anti-inflammatory cytokine IL-10. This article also reviews the literature supporting the key role that telomere loss plays in regard to increasing the ability of vertebrate (including placental) DNA to stimulate TLR9, and in regard to signaling the onset of apoptosis in the placenta and fetal membranes, thereby providing a biologic clock that determines the length of gestation and the timing for the onset of parturition. In summary, this literature review provides a strong rationale for future research to test the hypothesis that telomere loss and increased cffDNA levels trigger the proinflammatory events leading to the spontaneous onset of parturition in mammals: the "cffDNA/telomere hypothesis." PMID:26134037

  19. Cell-free fetal DNA and pregnancy-related complications (Review)

    Science.gov (United States)

    SIFAKIS, STAVROS; KOUKOU, ZETA; SPANDIDOS, DEMETRIOS A.

    2015-01-01

    Cell-free fetal DNA (cff-DNA) is a novel promising biomarker that has been applied in various aspects of obstetrical research, notably in prenatal diagnosis and complicated pregnancies. It is easily detected by semi-quantitative PCR for the SRY target gene. It is well recognized that the levels of circulating cff-DNA play a role in various complications of pregnancy. In this review, we explore the implications of the detection of cff-DNA in a range of pregnancy-related complications, such as preeclampsia, intrauterine growth restriction (IUGR), preterm labor, placenta previa and hyperemesis gravidarum. cff-DNA is released due to apoptotic mechanisms occurring on trophoblastic cells, although recent in vivo studies support the existence of additional mechanisms. The increase in the levels of cff-DNA can be used to predict pregnancy-related complications and has great value in the field of prenatal diagnosis and in common pregnancy-related complications, as it precedes the clinical symptoms of the disease. Gestational age is a factor that determines the elevation in cff-DNA levels in response to pathological conditions. In conclusion, the detection of cff-DNA levels has a number of valuable applications in prenatal screening; however, the detection of cff-DNA levels has not yet been applied in clinical practice for the diagnosis of pregnancy-related disorders. Thus, studies are focusing on unraveling the etiology of alterations in its levels under pathological conditions during pregnancy, in order to determine the potenial predictive and diagnostic applications of this biomarker. PMID:25530428

  20. Quantification of Maternal Serum Cell-Free Fetal DNA in Early-Onset Preeclampsia

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    Mulan Ren

    2013-04-01

    Full Text Available The aim of this study was to determine whether the increased serum cell-free fetal DNA (cffDNA level of gravidas developed into early-onset preeclampsia (EOPE subsequently in the early second trimesters is related to prenatal screening markers. Serum was collected from 1011 gravidas. The level of cffDNA and prenatal screening markers were analyzed in 20 cases with EOPE and 20 controls. All fetuses were male. The maternal serum cffDNA level was assessed by amplification of the Y chromosome specific gene. Correlations between the variables were examined. (Logged cffDNA in EOPE (median, 3.08; interquartile range, 2.93–3.68 was higher than controls (median, 1.79; interquartile range, 1.46–2.53. The increased level of (logged cffDNA was correlated significantly with the increased human chorionic gonadotropin (HCG level (r = 0.628, p < 0.001. Significant reciprocal correlations between cffDNA and babies’ birth weight as well as gestation weeks at delivery were noted (r = −0.516, p = 0.001; r = −0.623, p < 0.001, respectively. The sensitivity and specificity of cffDNA to discriminate between the EOPE cases and the controls were 90% and 85%, respectively. CffDNA is a potential marker for EOPE, which had a significant reciprocal correlation with babies’ birth weight and gestation weeks at delivery. Moreover, it may help in indicating the underlying hypoxic condition in the placenta.

  1. Cell-free fetal DNA in the maternal circulation originates from the cytotrophoblast: proof from an unique case

    Science.gov (United States)

    Hochstenbach, Ron; Nikkels, Peter G J; Elferink, Martin G; Oudijk, Martijn A; van Oppen, Carla; van Zon, Patrick; van Harssel, Jeske; Schuring-Blom, Heleen; Page-Christiaens, Godelieve C M L

    2015-01-01

    Key Clinical Message Noninvasive prenatal testing (NIPT) and direct karyotyping of cytotrophoblast were normal for a male fetus, but cultured chorionic villus mesenchymal cells and umbilical cord fibroblasts showed nonmosaic trisomy 18. This observation provides direct evidence for the cytotrophoblastic origin of cell-free fetal DNA and yields a biological explanation for falsely reassuring NIPT results. PMID:26185654

  2. Cell-free fetal DNA in the maternal circulation originates from the cytotrophoblast: proof from an unique case

    OpenAIRE

    Hochstenbach, Ron; Nikkels, Peter G. J.; Martin G Elferink; Oudijk, Martijn A; Oppen, Carla; van Zon, Patrick; van Harssel, Jeske; Schuring-Blom, Heleen; Page-Christiaens, Godelieve C M L

    2015-01-01

    Key Clinical Message Noninvasive prenatal testing (NIPT) and direct karyotyping of cytotrophoblast were normal for a male fetus, but cultured chorionic villus mesenchymal cells and umbilical cord fibroblasts showed nonmosaic trisomy 18. This observation provides direct evidence for the cytotrophoblastic origin of cell-free fetal DNA and yields a biological explanation for falsely reassuring NIPT results.

  3. Cell-free fetal DNA in the maternal circulation originates from the cytotrophoblast : proof from an unique case

    OpenAIRE

    Hochstenbach, Ron; Nikkels, Peter G. J.; Martin G Elferink; Oudijk, Martijn A; Oppen, Carla; van Zon, Patrick; van Harssel, Jeske; Schuring-Blom, Heleen; Page-Christiaens, Godelieve C M L

    2015-01-01

    Noninvasive prenatal testing (NIPT) and direct karyotyping of cytotrophoblast were normal for a male fetus, but cultured chorionic villus mesenchymal cells and umbilical cord fibroblasts showed nonmosaic trisomy 18. This observation provides direct evidence for the cytotrophoblastic origin of cell-free fetal DNA and yields a biological explanation for falsely reassuring NIPT results.

  4. Review: Cell-free fetal DNA in the maternal circulation as an indication of placental health and disease

    Science.gov (United States)

    Taglauer, E.S.; Wilkins-Haug, L.; Bianchi, D.W.

    2016-01-01

    In human pregnancy, the constant turnover of villous trophoblast results in extrusion of apoptotic material into the maternal circulation. This material includes cell-free (cf) DNA, which is commonly referred to as “fetal”, but is actually derived from the placenta. As the release of cf DNA is closely tied to placental morphogenesis, conditions associated with abnormal placentation, such as preeclampsia, are associated with high DNA levels in the blood of pregnant women. Over the past five years, the development and commercial availability of techniques of massively parallel DNA sequencing have facilitated noninvasive prenatal testing (NIPT) for fetal trisomies 13, 18, and 21. Clinical experience accrued over the past two years has highlighted the importance of the fetal fraction (ff) in cf DNA analysis. The ff is the amount of cell-free fetal DNA in a given sample divided by the total amount of cell-free DNA. At any gestational age, ff has a bell-shaped distribution that peaks between 10 and 20% at 10–21 weeks. ff is affected by maternal body mass index, gestational age, fetal aneuploidy, and whether the gestation is a singleton or multiple. In approximately 0.1% of clinical cases, the NIPT result and a subsequent diagnostic karyotype are discordant; confined placental mosaicism has been increasingly reported as an underlying biologic explanation. Cell-free fetal DNA is a new biomarker that can provide information about the placenta and potentially be used to predict clinical problems. Knowledge gaps still exist with regard to what affects production, metabolism, and clearance of feto-placental DNA. PMID:24388429

  5. Evaluation of a Modified DNA Extraction Method for Isolation of Cell-Free Fetal DNA from Maternal Serum

    Science.gov (United States)

    Keshavarz, Zeinab; Moezzi, Leili; Ranjbaran, Reza; Aboualizadeh, Farzaneh; Behzad-Behbahani, Abbas; Abdullahi, Masooma; Sharifzadeh, Sedigheh

    2015-01-01

    Background: Discovery of short cell free fetal DNA (cffDNA) fragments in maternal plasma has created major changes in the field of prenatal diagnosis. The use of cffDNA to set up noninvasive prenatal test is limited due to the low concentration of fetal DNA in maternal plasma therefore, employing a high efficiency extraction method leads to more accurate results. The aim of this study was to evaluate the efficiency of Triton/Heat/Phenol (THP) protocol in comparison with the QIAamp DNA Blood mini Kit for cffDNA purification. Methods: In order to evaluate the efficiency of THP protocol, DNA of Rhesus D (RhD) negative pregnant women's plasma was collected, then real-time PCR for RHD exon 7 was performed. The Ct value data of real time PCR obtained by two different methods were compared and after delivery serology test on cord blood was done to validate the real time PCR results. Results: The results indicated significant differences between two extraction methods (p=0.001). The mean±SD of Ct-value using THP protocol was 33.8±1.6 and 36.1±2.47 using QIAamp DNA Blood mini Kit. Conclusion: Our finding demonstrated that THP protocol was more effective than the QIAamp DNA Blood mini Kits for cffDNA extraction and lead to decrease the false negative results. PMID:26140187

  6. Fetal blood grouping using cell free DNA - an improved service for RhD negative pregnant women.

    Science.gov (United States)

    Bills, V L; Soothill, P W

    2014-04-01

    Red cell alloimmunisation involves the transplacental movement of maternally derived red cell antibodies into the fetal circulation, causing red cell haemolysis, fetal anaemia and ultimately fetal death. Current standard UK practice is to prevent sensitisation to the D antigen by administering anti-D at about 28 weeks' gestation to all RhD negative pregnancies. The determination of fetal blood group by non-invasive cell free fetal DNA testing offers an improved and more efficient service to RhD negative pregnant women and avoids the potential iatrogenic harm associated with standard practice. It also has significantly improved the management of women with red cell alloimunisation to D and other antigens. This review summarises the past and future management of red cell alloimmunisation during pregnancy and the impact of ffDNA tests. PMID:24679596

  7. Maternal Cell free DNA based screening for fetal microdeletion and the importance of careful diagnostic follow up

    OpenAIRE

    Yatsenko, Svetlana A.; Peters, David; Saller, Devereux; Chu, Tianjiao; Clemens, Michelle; Rajkovic, Aleksandar

    2015-01-01

    Background Noninvasive prenatal screening (NIPS) by next-generation sequencing of cell free DNA (cfDNA) in maternal plasma is used to screen for common aneuploidies such as trisomy 21, in high risk pregnancies. NIPS can identify fetal genomic microdeletions, however sensitivity and specificity have not been systematically evaluated. Commercial companies have begun to offer expanded panels including screening for common microdeletion syndromes such as 22q11.2 deletion (DiGeorge syndrome) witho...

  8. The usage and current approaches of cell free fetal DNA (cffDNA) as a prenatal diagnostic method in fetal aneuploidy screening

    OpenAIRE

    Hülya Erbaba; Gül Pınar

    2015-01-01

    Prenatal diagnosis of invasive and noninvasive tests can be done in a way (NIPT), but because of the invasive methods have risks of infection and abortion, diagnosing non-invasive procedure increasing day by day. One of the widespread cell free fetal DNA in maternal blood test (cffDNA) that is increasing in clinical use has been drawing attention. The incidence of aneuploidy chromosomal anomaly of the kind in which all live births; Trisomy 21 (Down Syndrome) 1/800, trisomy 13 (Patau syndrome)...

  9. The usage and current approaches of cell free fetal DNA (cffDNA as a prenatal diagnostic method in fetal aneuploidy screening

    Directory of Open Access Journals (Sweden)

    Hülya Erbaba

    2015-12-01

    Full Text Available Prenatal diagnosis of invasive and noninvasive tests can be done in a way (NIPT, but because of the invasive methods have risks of infection and abortion, diagnosing non-invasive procedure increasing day by day. One of the widespread cell free fetal DNA in maternal blood test (cffDNA that is increasing in clinical use has been drawing attention. The incidence of aneuploidy chromosomal anomaly of the kind in which all live births; Trisomy 21 (Down Syndrome 1/800, trisomy 13 (Patau syndrome 1 /10,000, trisomy 18 (Edwards syndrome is a form of 1/6000. Because of the high mortality and morbidity, it is vital that congenital anomalies should be diagnosed in prenatal period. Aneuploidy testing for high-risk pregnant women after the 10th week of pregnancy in terms of the blood sample is taken and free fetal DNA in maternal plasma is based on the measurement of the relative amount. Knowledge of the current criteria for use by healthcare professionals in the field test will allow the exclusion of maternal and fetal risks. In this study, it is aimed to demonstrate current international approaches related to the positive and negative sides of non-invasive that is one of the prenatal diagnostic methods of cffDNA test. J Clin Exp Invest 2015; 6 (4: 414-417

  10. DASAF: An R Package for Deep Sequencing-Based Detection of Fetal Autosomal Abnormalities from Maternal Cell-Free DNA

    Directory of Open Access Journals (Sweden)

    Baohong Liu

    2016-01-01

    Full Text Available Background. With the development of massively parallel sequencing (MPS, noninvasive prenatal diagnosis using maternal cell-free DNA is fast becoming the preferred method of fetal chromosomal abnormality detection, due to its inherent high accuracy and low risk. Typically, MPS data is parsed to calculate a risk score, which is used to predict whether a fetal chromosome is normal or not. Although there are several highly sensitive and specific MPS data-parsing algorithms, there are currently no tools that implement these methods. Results. We developed an R package, detection of autosomal abnormalities for fetus (DASAF, that implements the three most popular trisomy detection methods—the standard Z-score (STDZ method, the GC correction Z-score (GCCZ method, and the internal reference Z-score (IRZ method—together with one subchromosome abnormality identification method (SCAZ. Conclusions. With the cost of DNA sequencing declining and with advances in personalized medicine, the demand for noninvasive prenatal testing will undoubtedly increase, which will in turn trigger an increase in the tools available for subsequent analysis. DASAF is a user-friendly tool, implemented in R, that supports identification of whole-chromosome as well as subchromosome abnormalities, based on maternal cell-free DNA sequencing data after genome mapping.

  11. Prenatal screening for fetal aneuploidies with cell-free DNA in the general pregnancy population: a cost-effectiveness analysis

    Science.gov (United States)

    Fairbrother, Genevieve; Burigo, John; Sharon, Thomas; Song, Ken

    2016-01-01

    Abstract Objective: To estimate the cost-effectiveness of fetal aneuploidy screening in the general pregnancy population using non-invasive prenatal testing (NIPT) as compared to first trimester combined screening (FTS) with serum markers and NT ultrasound. Methods: Using a decision-analytic model, we estimated the number of fetal T21, T18, and T13 cases identified prenatally, the number of invasive procedures performed, corresponding normal fetus losses, and costs of screening using FTS or NIPT with cell-free DNA (cfDNA). Modeling was based on a 4 million pregnant women cohort, which represents annual births in the U.S. Results: For the general pregnancy population, NIPT identified 15% more trisomy cases, reduced invasive procedures by 88%, and reduced iatrogenic fetal loss by 94% as compared to FTS. The cost per trisomy case identified with FTS was $497 909. At a NIPT unit, cost of $453 and below, there were cost savings as compared to FTS. Accounting for additional trisomy cases identified by NIPT, a NIPT unit cost of $665 provided the same per trisomy cost as that of FTS. Conclusions: NIPT in the general pregnancy population leads to more prenatal identification of fetal trisomy cases as compared to FTS and is more economical at a NIPT unit cost of $453. PMID:26000626

  12. Fetal Aneuploidy Detection by Cell-Free DNA Sequencing for Multiple Pregnancies and Quality Issues with Vanishing Twins

    Directory of Open Access Journals (Sweden)

    Sebastian Grömminger

    2014-06-01

    Full Text Available Non-invasive prenatal testing (NIPT by random massively parallel sequencing of maternal plasma DNA for multiple pregnancies is a promising new option for prenatal care since conventional non-invasive screening for fetal trisomies 21, 18 and 13 has limitations and invasive diagnostic methods bear a higher risk for procedure related fetal losses in the case of multiple gestations compared to singletons. In this study, in a retrospective blinded analysis of stored twin samples, all 16 samples have been determined correctly, with four trisomy 21 positive and 12 trisomy negative samples. In the prospective part of the study, 40 blood samples from women with multiple pregnancies have been analyzed (two triplets and 38 twins, with two correctly identified trisomy 21 cases, confirmed by karyotyping. The remaining 38 samples, including the two triplet pregnancies, had trisomy negative results. However, NIPT is also prone to quality issues in case of multiple gestations: the minimum total amount of cell-free fetal DNA must be higher to reach a comparable sensitivity and vanishing twins may cause results that do not represent the genetics of the living sibling, as described in two case reports.

  13. Microarray-Based Analysis of Methylation Status of CpGs in Placental DNA and Maternal Blood DNA – Potential New Epigenetic Biomarkers for Cell Free Fetal DNA-Based Diagnosis

    OpenAIRE

    Hatt, Lotte; Aagaard, Mads M.; Graakjaer, Jesper; Bach, Cathrine; Sommer, Steffen; Inge E Agerholm; Kølvraa, Steen; Bojesen, Anders

    2015-01-01

    Epigenetic markers for cell free fetal DNA in the maternal blood circulation are highly interesting in the field of non-invasive prenatal testing since such markers will offer a possibility to quantify the amount of fetal DNA derived from different chromosomes in a maternal blood sample. The aim of the present study was to define new fetal specific epigenetic markers present in placental DNA that can be utilized in non-invasive prenatal diagnosis. We have conducted a high-resolution methylati...

  14. Prenatal diagnosis of Down syndrome using cell-free fetal DNA in amniotic fluid by quantitative fluorescent polymersase chain reaction

    Institute of Scientific and Technical Information of China (English)

    Wu Dan; Chi Hongbin; Shao Minjie; Wu Yao; Jin Hongyan; Wu Baiyan; Qiao Jie

    2014-01-01

    Backgroud Amniotic fluid (AF) supernatant contains cell-free fetal DNA (cffDNA) fragments.This study attempted to take advantage of cffDNA as a new material for prenatal diagnosis,which could be combined with simple quantitative fluorescent polymerase chain reaction (QF-PCR) to provide an ancillary method for the prenatal diagnosis of trisomy 21 syndrome.Methods AF supernatant samples were obtained from 27 women carrying euploid fetuses and 28 women carrying aneuploid fetuses with known cytogenetic karyotypes.Peripheral blood samples of the parents were collected at the same time.Short tandem repeat (STR) fragments on chromosome 21 were amplified by QF-PCR.Fetal condition and the parental source of the extra chromosome could be determined by the STR peaks.Results The sensitivity of the assay for the aneuploid was 93% (26/28; confidence interval,CI:77%-98%) and the specificity was 100% (26/26; CI:88%-100%).The determination rate of the origin of the extra chromosome was 69%.The sensitivity and the specificity of the assay in the euploid were 100% (27/27).Conclusions Trisomy 21 can be prenatally diagnosed by the QF-PCR method in AF supernatant.This karyotype analysis method greatly reduces the requirement for the specimen size.It will be a benefit for early amniocentesis and could avoid pregnancy complications.The method may become an ancillary method for prenatal diagnosis of trisomy 21.

  15. Early Fetal Gender Determination Using Real-Time PCR Analysis of Cell-free Fetal DNA During 6th-10th Weeks of Gestation

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    Hamid Reza Khorram Khorshid

    2013-04-01

    Full Text Available Nowadays, new advances in the use of cell free fetal DNA (cffDNA in maternal plasma of pregnant women has provided the possibility of applying cffDNA in prenatal diagnosis as a non-invasive method. In contrary to the risks of invasive methods that affect both mother and fetus, applying cffDNA is proven to be highly effective with lower risk. One of the applications of prenatal diagnosis is fetal gender determination, which is important in fetuses at risk of sex-linked genetic diseases. In such cases by obtaining the basic information of the gender, necessary time management can be taken in therapeutic to significantly reduce the necessity of applying the invasive methods. Therefore in this study, the probability of detecting sequences on the human Y-chromosome in pregnant women has been evaluated to identify the gender of fetuses. Peripheral blood samples were obtained from 80 pregnant women with gestational age between 6th to 10th weeks and the fetal DNA was extracted from the plasma. Identification of SRY, DYS14 & DAZ sequences, which are not presentin the maternal genome, was performed using Real-Time PCR. All the obtained results were compared with the actual gender of the newborns to calculate the test accuracy. Considerable 97.3% sensitivity and 97.3% specificity were obtained in fetal gender determination which is significant in the first trimester of pregnancy. Only in one case, false positive result was obtained. Using non-invasive method of cffDNAs in the shortest time possible, as well as avoiding invasive tests for early determination of fetal gender, provides the opportunity of deciding and employing early treatment for fetuses at risk of genetic diseases

  16. Non-Invasive Prenatal RHD Genotyping Using Cell-Free Fetal DNA from Maternal Plasma: An Italian Experience

    Science.gov (United States)

    Picchiassi, Elena; Di Renzo, Gian Carlo; Tarquini, Federica; Bini, Vittorio; Centra, Michela; Pennacchi, Luana; Galeone, Fabiana; Micanti, Mara; Coata, Giuliana

    2015-01-01

    Summary Background This study assessed the diagnostic accuracy of a non-invasive approach to fetal RHD genotyping using cell-free fetal DNA in maternal plasma and a combination of methodological strategies. Methods Real-time PCR (qPCR) was performed on 216 RhD-negative women between weeks 10+0 and 14+6 of gestation (1st qPCR). qPCR was repeated (2nd qPCR) to increase the amount of each sample for analysis, on 95 plasma aliquots that were available from first trimester blood collection (group 1) and on 13 samples that were collected between weeks 18+0 and 25+6 of gestation (group 2). qPCR was specific for exons 5 and 7 of the RHD gene (RHD5 and RHD7). The results were interpreted according to the number of positive replicates of both exons. Results 1st qPCR: diagnostic accuracy was of 93.3%. Diagnostic accuracy increased from 90.5% (1st qPCR) to 93.7% (2nd qPCR) in group 1 and from 84.6% (1st qPCR) to 92.3% (2nd qPCR) in group 2. These increments were not statistically significant. Conclusion Our approach to RHD genotyping in early pregnancy yielded high diagnostic accuracy. Increasing the amount of DNA analyzed in each sample did not improve significantly the diagnostic accuracy of the test. PMID:25960712

  17. A Method to Quantify Cell-Free Fetal DNA Fraction in Maternal Plasma Using Next Generation Sequencing: Its Application in Non-Invasive Prenatal Chromosomal Aneuploidy Detection

    OpenAIRE

    Xu, Xu-Ping; Gan, Hai-Yan; Li, Fen-xia; Tian, Qi; Zhang, Jun; Liang, Rong-Liang; LI Ming; Yang, Xue-Xi; Wu, Ying-Song

    2016-01-01

    Objective The fraction of circulating cell-free fetal (cff) DNA in maternal plasma is a critical parameter for aneuploidy screening with non-invasive prenatal testing, especially for those samples located in equivocal zones. We developed an approach to quantify cff DNA fractions directly with sequencing data, and increased cff DNAs by optimizing library construction procedure. Methods Artificial DNA mixture samples (360), with known cff DNA fractions, were used to develop a method to determin...

  18. Evaluation of Sample Stability and Automated DNA Extraction for Fetal Sex Determination Using Cell-Free Fetal DNA in Maternal Plasma

    OpenAIRE

    Elena Ordoñez; Laura Rueda; M. Paz Cañadas; Carme Fuster; Vincenzo Cirigliano

    2013-01-01

    Objective. The detection of paternally inherited sequences in maternal plasma, such as the SRY gene for fetal sexing or RHD for fetal blood group genotyping, is becoming part of daily routine in diagnostic laboratories. Due to the low percentage of fetal DNA, it is crucial to ensure sample stability and the efficiency of DNA extraction. We evaluated blood stability at 4°C for at least 24 hours and automated DNA extraction, for fetal sex determination in maternal plasma. Methods. A total of 15...

  19. Noninvasive prenatal diagnosis of fetal RhD status using cell-free fetal DNA in maternal plasma with TaqMan® real-time PCR assay

    OpenAIRE

    Rekhviashvili, Tea

    2007-01-01

    Prenatal diagnosis is now part of established obstetric practice in many countries. However, conventional methods of prenatal diagnosis of obtaining fetal tissues for genetic analysis, including amniocentesis and chorionic villus sampling, are invasive and constitute a finite risk to the unborn fetus1. At present, it is widely accepted that both intact fetal cells as well as cell-free fetal DN A are present in the maternal circulation and can be recovered for non-invasive prena...

  20. Evaluation of Sample Stability and Automated DNA Extraction for Fetal Sex Determination Using Cell-Free Fetal DNA in Maternal Plasma

    Directory of Open Access Journals (Sweden)

    Elena Ordoñez

    2013-01-01

    Full Text Available Objective. The detection of paternally inherited sequences in maternal plasma, such as the SRY gene for fetal sexing or RHD for fetal blood group genotyping, is becoming part of daily routine in diagnostic laboratories. Due to the low percentage of fetal DNA, it is crucial to ensure sample stability and the efficiency of DNA extraction. We evaluated blood stability at 4°C for at least 24 hours and automated DNA extraction, for fetal sex determination in maternal plasma. Methods. A total of 158 blood samples were collected, using EDTA-K tubes, from women in their 1st trimester of pregnancy. Samples were kept at 4°C for at least 24 hours before processing. An automated DNA extraction was evaluated, and its efficiency was compared with a standard manual procedure. The SRY marker was used to quantify cfDNA by real-time PCR. Results. Although lower cfDNA amounts were obtained by automated DNA extraction (mean 107,35 GE/mL versus 259,43 GE/mL, the SRY sequence was successfully detected in all 108 samples from pregnancies with male fetuses. Conclusion. We successfully evaluated the suitability of standard blood tubes for the collection of maternal blood and assessed samples to be suitable for analysis at least 24 hours later. This would allow shipping to a central reference laboratory almost from anywhere in Europe.

  1. Detection of fetal cell-free DNA in maternal plasma for Down syndrome, Edward syndrome and Patau syndrome of high risk fetus

    OpenAIRE

    Ke, Wei-Lin; Zhao, Wei-Hua; Wang, Xin-Yu

    2015-01-01

    Objective: The study aimed to validate the efficacy of detection of fetal cell-free DNA in maternal plasma of trisomy 21, 18 and 13 in a clinical setting. Methods: A total of 2340 women at high risk for Down syndrome based on maternal age, prenatal history or a positive sesum or sonographic screening test were offered prenatal noninvasive aneuploidy test. According to the prenatal noninvasive aneuploidy test, the pregnant women at high risk were offered amniocentesis karyotype analysis and th...

  2. A Method to Quantify Cell-Free Fetal DNA Fraction in Maternal Plasma Using Next Generation Sequencing: Its Application in Non-Invasive Prenatal Chromosomal Aneuploidy Detection.

    Directory of Open Access Journals (Sweden)

    Xu-Ping Xu

    Full Text Available The fraction of circulating cell-free fetal (cff DNA in maternal plasma is a critical parameter for aneuploidy screening with non-invasive prenatal testing, especially for those samples located in equivocal zones. We developed an approach to quantify cff DNA fractions directly with sequencing data, and increased cff DNAs by optimizing library construction procedure.Artificial DNA mixture samples (360, with known cff DNA fractions, were used to develop a method to determine cff DNA fraction through calculating the proportion of Y chromosomal unique reads, with sequencing data generated by Ion Proton. To validate our method, we investigated cff DNA fractions of 2,063 pregnant women with fetuses who were diagnosed as high risk of fetal defects. The z-score was calculated to determine aneuploidies for chromosomes 21, 18 and 13. The relationships between z-score and parameters of pregnancies were also analyzed. To improve cff DNA fractions in our samples, two groups were established as follows: in group A, the large-size DNA fragments were removed, and in group B these were retained, during library construction.A method to determine cff DNA fractions was successfully developed using 360 artificial mixture samples in which cff DNA fractions were known. A strong positive correlation was found between z-score and fetal DNA fraction in the artificial mixture samples of trisomy 21, 18 and 13, as well as in clinical maternal plasma samples. There was a positive correlation between gestational age and the cff DNA fraction in the clinical samples, but no correlation for maternal age. Moreover, increased fetal DNA fractions were found in group A compared to group B.A relatively accurate method was developed to determine the cff DNA fraction in maternal plasma. By optimizing, we can improve cff DNA fractions in sequencing samples, which may contribute to improvements in detection rate and reliability.

  3. A Method to Quantify Cell-Free Fetal DNA Fraction in Maternal Plasma Using Next Generation Sequencing: Its Application in Non-Invasive Prenatal Chromosomal Aneuploidy Detection

    Science.gov (United States)

    Xu, Xu-Ping; Gan, Hai-Yan; Li, Fen-Xia; Tian, Qi; Zhang, Jun; Liang, Rong-Liang; Li, Ming

    2016-01-01

    Objective The fraction of circulating cell-free fetal (cff) DNA in maternal plasma is a critical parameter for aneuploidy screening with non-invasive prenatal testing, especially for those samples located in equivocal zones. We developed an approach to quantify cff DNA fractions directly with sequencing data, and increased cff DNAs by optimizing library construction procedure. Methods Artificial DNA mixture samples (360), with known cff DNA fractions, were used to develop a method to determine cff DNA fraction through calculating the proportion of Y chromosomal unique reads, with sequencing data generated by Ion Proton. To validate our method, we investigated cff DNA fractions of 2,063 pregnant women with fetuses who were diagnosed as high risk of fetal defects. The z-score was calculated to determine aneuploidies for chromosomes 21, 18 and 13. The relationships between z-score and parameters of pregnancies were also analyzed. To improve cff DNA fractions in our samples, two groups were established as follows: in group A, the large-size DNA fragments were removed, and in group B these were retained, during library construction. Results A method to determine cff DNA fractions was successfully developed using 360 artificial mixture samples in which cff DNA fractions were known. A strong positive correlation was found between z-score and fetal DNA fraction in the artificial mixture samples of trisomy 21, 18 and 13, as well as in clinical maternal plasma samples. There was a positive correlation between gestational age and the cff DNA fraction in the clinical samples, but no correlation for maternal age. Moreover, increased fetal DNA fractions were found in group A compared to group B. Conclusion A relatively accurate method was developed to determine the cff DNA fraction in maternal plasma. By optimizing, we can improve cff DNA fractions in sequencing samples, which may contribute to improvements in detection rate and reliability. PMID:26765738

  4. Non-invasive prenatal diagnosis of β-thalassemia by detection of the cell-free fetal DNA in maternal circulation: a systematic review and meta-analysis.

    Science.gov (United States)

    Zafari, Mandana; Kosaryan, Mehrnoush; Gill, Pooria; Alipour, Abbass; Shiran, Mohammadreza; Jalalli, Hossein; Banihashemi, Ali; Fatahi, Fatemeh

    2016-08-01

    The discovery of fetal DNA (f-DNA) opens the possibility of early non-invasive procedure for detection of paternally inherited mutation of beta-thalassemia. Since 2002, some studies have examined the sensitivity and specificity of this method for detection of paternally inherited mutation of thalassemia in pregnant women at risk of having affected babies. We conducted a systematic review of published articles that evaluated using this method for early detection of paternally inherited mutation in maternal plasma. A sensitive search of multiple databases was done in which nine studies met our inclusion criteria. The sensitivity and specificity was 99 and 99 %, respectively. The current study found that detection of paternally inherited mutation of thalassemia using analysis of cell-free fetal DNA is highly accurate. This method could replace conventional and invasive methods. PMID:26968552

  5. Non-invasive prenatal diagnostic test accuracy for fetal sex using cell-free DNA a review and meta-analysis

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    Wright Caroline F

    2012-09-01

    Full Text Available Abstract Background Cell-free fetal DNA (cffDNA can be detected in maternal blood during pregnancy, opening the possibility of early non-invasive prenatal diagnosis for a variety of genetic conditions. Since 1997, many studies have examined the accuracy of prenatal fetal sex determination using cffDNA, particularly for pregnancies at risk of an X-linked condition. Here we report a review and meta-analysis of the published literature to evaluate the use of cffDNA for prenatal determination (diagnosis of fetal sex. We applied a sensitive search of multiple bibliographic databases including PubMed (MEDLINE, EMBASE, the Cochrane library and Web of Science. Results Ninety studies, incorporating 9,965 pregnancies and 10,587 fetal sex results met our inclusion criteria. Overall mean sensitivity was 96.6% (95% credible interval 95.2% to 97.7% and mean specificity was 98.9% (95% CI = 98.1% to 99.4%. These results vary very little with trimester or week of testing, indicating that the performance of the test is reliably high. Conclusions Based on this review and meta-analysis we conclude that fetal sex can be determined with a high level of accuracy by analyzing cffDNA. Using cffDNA in prenatal diagnosis to replace or complement existing invasive methods can remove or reduce the risk of miscarriage. Future work should concentrate on the economic and ethical considerations of implementing an early non-invasive test for fetal sex.

  6. Microarray-Based Analysis of Methylation Status of CpGs in Placental DNA and Maternal Blood DNA - Potential New Epigenetic Biomarkers for Cell Free Fetal DNA-Based Diagnosis

    DEFF Research Database (Denmark)

    Hatt, Lotte; Aagaard, Mads M; Graakjaer, Jesper;

    2015-01-01

    Epigenetic markers for cell free fetal DNA in the maternal blood circulation are highly interesting in the field of non-invasive prenatal testing since such markers will offer a possibility to quantify the amount of fetal DNA derived from different chromosomes in a maternal blood sample. The aim of...... the present study was to define new fetal specific epigenetic markers present in placental DNA that can be utilized in non-invasive prenatal diagnosis. We have conducted a high-resolution methylation specific beadchip microarray study assessing more than 450.000 CpG sites. We have analyzed the DNA...... with a potential for aneuploidy testing as well as a list of markers for regions harboring sub-microscopic deletion- or duplication syndromes....

  7. Impact of Cell-Free Fetal DNA Screening on Patients’ Choice of Invasive Procedures after a Positive California Prenatal Screen Result

    Directory of Open Access Journals (Sweden)

    Forum T. Shah

    2014-07-01

    Full Text Available Until recently, maternal serum analyte levels paired with sonographic fetal nuchal translucency measurement was the most accurate prenatal screen available for Trisomies 18 and 21, (91% and 94% detection and false positive rates of 0.31% and 4.5% respectively. Women with positive California Prenatal Screening Program (CPSP results have the option of diagnostic testing to determine definitively if the fetus has a chromosomal abnormality. Cell-free fetal (cff- DNA screening for Trisomies 13, 18, and 21 was first offered in 2012, allowing women with positive screens to choose additional screening before diagnostic testing. Cff-DNA sensitivity rates are as high as 99.9% and 99.1%, with false positive rates of 0.4% and 0.1%, for Trisomies 18 and 21, respectively. A retrospective chart review was performed in 2012 on 500 CPSP referrals at the University of California, San Diego Thornton Hospital. Data were collected prior to and after the introduction of cff-DNA. There was a significant increase in the number of participants who chose to pursue additional testing and a decrease in the number of invasive procedures performed after cff-DNA screening was available. We conclude that as fetal aneuploidy screening improves, the number of invasive procedures will continue to decrease.

  8. Pre-Analytical Conditions in Non-Invasive Prenatal Testing of Cell-Free Fetal RHD

    DEFF Research Database (Denmark)

    Clausen, Frederik Banch; Jakobsen, Tanja Roien; Rieneck, Klaus;

    2013-01-01

    Non-invasive prenatal testing of cell-free fetal DNA (cffDNA) in maternal plasma can predict the fetal RhD type in D negative pregnant women. In Denmark, routine antenatal screening for the fetal RhD gene (RHD) directs the administration of antenatal anti-D prophylaxis only to women who carry an Rh...

  9. Identifying mild and severe preeclampsia in asymptomatic pregnant women by levels of cell-free fetal DNA

    DEFF Research Database (Denmark)

    Jakobsen, Tanja Roien; Clausen, Frederik Banch; Rode, Line;

    2013-01-01

    BACKGROUND: The objective was to investigate whether women who develop preeclampsia can be identified in a routine analysis when determining fetal RHD status at 25 weeks' gestation in combination with PAPP-A levels at the first-trimester combined risk assessment for Trisomy 21. STUDY DESIGN AND...... preeclampsia, but adding it to the analysis did not increase the detection rate (DR). CONCLUSION: Women with cffDNA levels below the 5th percentile and above the 90th percentile quantified at 25 weeks' gestation are at increased risk of developing preeclampsia. Adding PAPP-A levels to the analysis did not...

  10. A Non-Invasive Droplet Digital PCR (ddPCR) Assay to Detect Paternal CFTR Mutations in the Cell-Free Fetal DNA (cffDNA) of Three Pregnancies at Risk of Cystic Fibrosis via Compound Heterozygosity

    OpenAIRE

    Debrand, Emmanuel; Lykoudi, Alexandra; Bradshaw, Elizabeth; Allen, Stephanie K.

    2015-01-01

    Introduction Non-invasive prenatal diagnosis (NIPD) makes use of cell-free fetal DNA (cffDNA) in the mother’s bloodstream as an alternative to invasive sampling methods such as amniocentesis or CVS, which carry a 0.5–1% risk of fetal loss. We describe a droplet digital PCR (ddPCR) assay designed to inform the testing options for couples whose offspring are at risk of suffering from cystic fibrosis via compound heterozygosity. By detecting the presence or absence of the paternal mutation in th...

  11. Positive predictive value of non-invasive prenatal screening for fetal chromosome disorders using cell-free DNA in maternal serum: independent clinical experience of a tertiary referral center

    OpenAIRE

    Neufeld-Kaiser, Whitney A.; Cheng, Edith Y.; Liu, Yajuan J

    2015-01-01

    Background Non-invasive prenatal screening (NIPS) for fetal chromosome abnormalities using cell-free deoxyribonucleic acid (cfDNA) in maternal serum has significantly influenced prenatal diagnosis of fetal aneuploidies since becoming clinically available in the fall of 2011. High sensitivity and specificity have been reported in multiple publications, nearly all of which have been sponsored by the commercial performing laboratories. Once results are returned, positive and negative predictive ...

  12. Fetal RHD Genotyping Using Real-Time Polymerase Chain Reaction Analysis of Cell-Free Fetal DNA in Pregnancy of RhD Negative Women in South of Iran

    Directory of Open Access Journals (Sweden)

    Leili Moezzi

    2016-05-01

    Full Text Available Background: Maternal-fetal RhD antigen incompatibility causes approximately 50% of clinically significant alloimmunization cases. The routine use of prophylactic anti-D immunoglobulin has dramatically reduced hemolytic disease of the fetus and newborn. Recently, fetal RHD genotyping in RhD negative pregnant women has been suggested for appropriate use of anti-D immunoglobulin antenatal prophylaxis and decrease unnecessary prenatal interventions. Materials and Methods: In this prospective cohort study, in order to develop a reliable and non-invasive method for fetal RHD genotyping, cell free fetal DNA (cffDNA was extracted from maternal plasma. Real-time quantitative polymerase chain reaction (qPCR for detection of RHD exons 7, 5, 10 and intron 4 was performed and the results were compared to the serological results of cord blood cells as the gold standard method. SRY gene and hypermethylated Ras-association domain family member 1 (RASSF1A gene were used to confirm the presence of fetal DNA in male and female fetuses, respectively. Results: Out of 48 fetuses between 8 and 32 weeks (wks of gestational age (GA, we correctly diagnosed 45 cases (93.75% of RHD positive fetuses and 2 cases (4.16% of the RHD negative one. Exon 7 was amplified in one sample, while three other RHD gene sequences were not detected; the sample was classified as inconclusive, and the RhD serology result after birth showed that the fetus was RhD-negative. Conclusion: Our results showed high accuracy of the qPCR method using cffDNA for fetal RHD genotyping and implicate on the efficiency of this technique to predict the competence of anti-D immunoglobulin administration.

  13. Prenatal screening for fetal aneuploidies with cell-free DNA in the general pregnancy population: a cost-effectiveness analysis

    OpenAIRE

    Fairbrother, Genevieve; Burigo, John; Sharon, Thomas; Song, Ken

    2015-01-01

    Abstract Objective: To estimate the cost-effectiveness of fetal aneuploidy screening in the general pregnancy population using non-invasive prenatal testing (NIPT) as compared to first trimester combined screening (FTS) with serum markers and NT ultrasound. Methods: Using a decision-analytic model, we estimated the number of fetal T21, T18, and T13 cases identified prenatally, the number of invasive procedures performed, corresponding normal fetus losses, and costs of screening using FTS or N...

  14. Fetal RHD Genotyping Using Real-Time Polymerase Chain Reaction Analysis of Cell-Free Fetal DNA in Pregnancy of RhD Negative Women in South of Iran

    Science.gov (United States)

    Moezzi, Leili; Keshavarz, Zeinab; Ranjbaran, Reza; Aboualizadeh, Farzaneh; Behzad-Behbahani, Abbas; Abdullahi, Masooma; Ramezani, Amin; Samsami, Alamtaj; Sharifzadeh, Sedigheh

    2016-01-01

    Background Maternal-fetal RhD antigen incompatibility causes approximately 50% of clinically significant alloimmunization cases. The routine use of prophylactic anti-D immunoglobulin has dramatically reduced hemolytic disease of the fetus and newborn. Recently, fetal RHD genotyping in RhD negative pregnant women has been suggested for appropriate use of anti-D immunoglobulin antenatal prophylaxis and decrease unnecessary prenatal interventions. Materials and Methods In this prospective cohort study, in order to develop a reliable and non-invasive method for fetal RHD genotyping, cell free fetal DNA (cffD- NA) was extracted from maternal plasma. Real-time quantitative polymerase chain reaction (qPCR) for detection of RHD exons 7, 5, 10 and intron 4 was performed and the results were compared to the serological results of cord blood cells as the gold standard method. SRY gene and hypermethylated Ras-association domain family member 1 (RASSF1A) gene were used to confirm the presence of fetal DNA in male and female fetuses, respectively. Results Out of 48 fetuses between 8 and 32 weeks (wks) of gestational age (GA), we correctly diagnosed 45 cases (93.75%) of RHD positive fetuses and 2 cases (4.16%) of the RHD negative one. Exon 7 was amplified in one sample, while three other RHD gene sequences were not detected; the sample was classified as inconclusive, and the RhD serology result after birth showed that the fetus was RhD-negative. Conclusion Our results showed high accuracy of the qPCR method using cffDNA for fetal RHD genotyping and implicate on the efficiency of this technique to predict the competence of anti-D immunoglobulin administration.

  15. Microarray-Based Analysis of Methylation Status of CpGs in Placental DNA and Maternal Blood DNA--Potential New Epigenetic Biomarkers for Cell Free Fetal DNA-Based Diagnosis.

    Directory of Open Access Journals (Sweden)

    Lotte Hatt

    Full Text Available Epigenetic markers for cell free fetal DNA in the maternal blood circulation are highly interesting in the field of non-invasive prenatal testing since such markers will offer a possibility to quantify the amount of fetal DNA derived from different chromosomes in a maternal blood sample. The aim of the present study was to define new fetal specific epigenetic markers present in placental DNA that can be utilized in non-invasive prenatal diagnosis. We have conducted a high-resolution methylation specific beadchip microarray study assessing more than 450.000 CpG sites. We have analyzed the DNA methylation profiles of 10 maternal blood samples and compared them to 12 1st trimesters chorionic samples from normal placentas, identifying a number of CpG sites that are differentially methylated in maternal blood cells compared to chorionic tissue. To strengthen the utility of these differentially methylated CpG sites to be used with methyl-sensitive restriction enzymes (MSRE in PCR-based NIPD, we furthermore refined the list of selected sites, containing a restriction sites for one of 16 different methylation-sensitive restriction enzymes. We present a list of markers on chromosomes 13, 18 and 21 with a potential for aneuploidy testing as well as a list of markers for regions harboring sub-microscopic deletion- or duplication syndromes.

  16. Detection of fetal cell-free DNA in maternal plasma for Down syndrome, Edward syndrome and Patau syndrome of high risk fetus

    Science.gov (United States)

    Ke, Wei-Lin; Zhao, Wei-Hua; Wang, Xin-Yu

    2015-01-01

    Objective: The study aimed to validate the efficacy of detection of fetal cell-free DNA in maternal plasma of trisomy 21, 18 and 13 in a clinical setting. Methods: A total of 2340 women at high risk for Down syndrome based on maternal age, prenatal history or a positive sesum or sonographic screening test were offered prenatal noninvasive aneuploidy test. According to the prenatal noninvasive aneuploidy test, the pregnant women at high risk were offered amniocentesis karyotype analysis and the pregnant at low risk were followed up to make sure the newborn outcome. Results: The prenatal noninvasive aneuploidy test was positive for trisomy 21 in 17 cases, for trisomy 18 in 6 cases and for trisomy 13 in 1 case, which of all were confirmed by karyotype analysis. Newborns of low risk gestational woman detected by prenatal noninvasive aneuploidy for trisomy 21, 18, 13 were followed up and no one was found with trisomy. Conclusions: The prenatal noninvasive aneuploidy test is highly accurate for detection of trisomy 21, 18 and 13, which can be considered as a practical alternative for traditional invasive diagnostic procedures. PMID:26309618

  17. Cell-free DNA: Comparison of Technologies.

    Science.gov (United States)

    Dar, Pe'er; Shani, Hagit; Evans, Mark I

    2016-06-01

    Cell-free fetal DNA screening for Down syndrome has gained rapid acceptance over the past few years with increasing market penetration. Three main laboratory methodologies are currently used: a massive parallel shotgun sequencing (MPSS), a targeted massive parallel sequencing (t-MPS) and a single nucleotide polymorphism (SNP) based approach. Although each of these technologies has its own advantages and disadvantages, the performance of all was shown to be comparable and superior to that of traditional first-trimester screening for the detection of trisomy 21 in a routine prenatal population. Differences in performance were predominantly shown for chromosomal anomalies other than trisomy 21. Understanding the limitations and benefits of each technology is essential for proper counseling to patients. These technologies, as well as few investigational technologies described in this review, carry a great potential beyond screening for the common aneuploidies. PMID:27235906

  18. Whole genome bisulfite sequencing of cell-free DNA and its cellular contributors uncovers placenta hypomethylated domains

    OpenAIRE

    Jensen, Taylor J.; Kim, Sung K; Zhu, Zhanyang; Chin, Christine; Gebhard, Claudia; Lu, Tim; Deciu, Cosmin; Van den Boom, Dirk; Ehrich, Mathias

    2015-01-01

    Background Circulating cell-free fetal DNA has enabled non-invasive prenatal fetal aneuploidy testing without direct discrimination of the maternal and fetal DNA. Testing may be improved by specifically enriching the sample material for fetal DNA. DNA methylation may allow for such a separation of DNA; however, this depends on knowledge of the methylomes of circulating cell-free DNA and its cellular contributors. Results We perform whole genome bisulfite sequencing on a set of unmatched sampl...

  19. Mrassf1a-pap, a novel methylation-based assay for the detection of cell-free fetal DNA in maternal plasma.

    Directory of Open Access Journals (Sweden)

    Jessica M E van den Oever

    Full Text Available OBJECTIVES: RASSF1A has been described to be differentially methylated between fetal and maternal DNA and can therefore be used as a universal sex-independent marker to confirm the presence of fetal sequences in maternal plasma. However, this requires highly sensitive methods. We have previously shown that Pyrophosphorolysis-activated Polymerization (PAP is a highly sensitive technique that can be used in noninvasive prenatal diagnosis. In this study, we have used PAP in combination with bisulfite conversion to develop a new universal methylation-based assay for the detection of fetal methylated RASSF1A sequences in maternal plasma. METHODS: Bisulfite sequencing was performed on maternal genomic (gDNA and fetal gDNA from chorionic villi to determine differentially methylated regions in the RASSF1A gene using bisulfite specific PCR primers. Methylation specific primers for PAP were designed for the detection of fetal methylated RASSF1A sequences after bisulfite conversion and validated. RESULTS: Serial dilutions of fetal gDNA in a background of maternal gDNA show a relative percentage of ~3% can be detected using this assay. Furthermore, fetal methylated RASSF1A sequences were detected both retrospectively as well as prospectively in all maternal plasma samples tested (n = 71. No methylated RASSF1A specific bands were observed in corresponding maternal gDNA. Specificity was further determined by testing anonymized plasma from non-pregnant females (n = 24 and males (n = 21. Also, no methylated RASSF1A sequences were detected here, showing this assay is very specific for methylated fetal DNA. Combining all samples and controls, we obtain an overall sensitivity and specificity of 100% (95% CI 98.4%-100%. CONCLUSIONS: Our data demonstrate that using a combination of bisulfite conversion and PAP fetal methylated RASSF1A sequences can be detected with extreme sensitivity in a universal and sex-independent manner. Therefore, this assay could be of great

  20. Model-based analysis of costs and outcomes of non-invasive prenatal testing for Down's syndrome using cell free fetal DNA in the UK National Health Service.

    Directory of Open Access Journals (Sweden)

    Stephen Morris

    Full Text Available BACKGROUND: Non-invasive prenatal testing (NIPT for Down's syndrome (DS using cell free fetal DNA in maternal blood has the potential to dramatically alter the way prenatal screening and diagnosis is delivered. Before NIPT can be implemented into routine practice, information is required on its costs and benefits. We investigated the costs and outcomes of NIPT for DS as contingent testing and as first-line testing compared with the current DS screening programme in the UK National Health Service. METHODS: We used a pre-existing model to evaluate the costs and outcomes associated with NIPT compared with the current DS screening programme. The analysis was based on a hypothetical screening population of 10,000 pregnant women. Model inputs were taken from published sources. The main outcome measures were number of DS cases detected, number of procedure-related miscarriages and total cost. RESULTS: At a screening risk cut-off of 1∶150 NIPT as contingent testing detects slightly fewer DS cases, has fewer procedure-related miscarriages, and costs the same as current DS screening (around UK£280,000 at a cost of £500 per NIPT. As first-line testing NIPT detects more DS cases, has fewer procedure-related miscarriages, and is more expensive than current screening at a cost of £50 per NIPT. When NIPT uptake increases, NIPT detects more DS cases with a small increase in procedure-related miscarriages and costs. CONCLUSIONS: NIPT is currently available in the private sector in the UK at a price of £400-£900. If the NHS cost was at the lower end of this range then at a screening risk cut-off of 1∶150 NIPT as contingent testing would be cost neutral or cost saving compared with current DS screening. As first-line testing NIPT is likely to produce more favourable outcomes but at greater cost. Further research is needed to evaluate NIPT under real world conditions.

  1. 孕DOWN综合征胎儿妇女血浆游离胎儿DNA浓度定量研究%Quantitative analysis of cell-free fetal DNA levels in maternal plasma with DOWN syndrome and normal pregnancies

    Institute of Scientific and Technical Information of China (English)

    孙建民; 谈月娣

    2009-01-01

    Objective To investigate the application of real-time quantitative PCR in quantification of cell-free fetal DNA maternal plasma in patients bearing fetuses affected with DOWN syndrome. Methods Cell-free fetal DNA in maternal serum was isolated from 30 samples(7 male DOWN syndrome fetal ,3 female DOWN syndrome fe-tal,14 male euploid fetal,6 female euploid fetal). Cell-free fetal DNA levels in maternal serum were measured using real-time quantitative PCR using SRY as marker. Results The median cell-free fetal DNA levels in pregnant carry-ing male fetuses(n=7) and the controls (pregnant carrying male euploid fetuses,n=14)were 318.03±96.74 ge-nome-equivalents/ml and 154.40±39.43 genome-equivalents/ml of maternal serum,respectively (t=3.33,P=0.004 ),which was o in women with female fetuses. Conclusion The cell-free fetal DNA levels in pregnant women with DOWN syndrome fetuses are higher than that in pregnant women with normal fetuses.%目的 探讨实时定量PCR方法检测妊娠DOWN综合征胎儿妇女血浆游离胎儿DNA的可行性.方法 SRY基因为胎儿游离DNA的标志,应用实时定量PCR分别检测10例妊娠DOWN综合征胎儿(男胎7例,女胎3例)妇女与20例妊娠正常胎儿(男胎14例,女胎6例)妇女血浆标本中游离胎儿DNA含量.结果 7例妊娠DOWN综合征男胎妇女SRY基因当量为(318.03±96.74)拷贝/ml(95%可信区间228.26~407.50拷贝/ml),妊娠正常男胎组SRY基因当量为(154.40±39.43)拷贝/ml(95%可信区间131.63~177.16拷贝/ml);2组差异有统计学意义(t=3.33,P=0.004).妊娠女胎组妇女血浆中SRY基因当量均为0拷贝/ml.结论 妊娠DOWN综合征男胎儿妇女血浆游离胎儿DNA的量较妊娠正常男胎儿妇女高.

  2. A Non-Invasive Droplet Digital PCR (ddPCR Assay to Detect Paternal CFTR Mutations in the Cell-Free Fetal DNA (cffDNA of Three Pregnancies at Risk of Cystic Fibrosis via Compound Heterozygosity.

    Directory of Open Access Journals (Sweden)

    Emmanuel Debrand

    Full Text Available Non-invasive prenatal diagnosis (NIPD makes use of cell-free fetal DNA (cffDNA in the mother's bloodstream as an alternative to invasive sampling methods such as amniocentesis or CVS, which carry a 0.5-1% risk of fetal loss. We describe a droplet digital PCR (ddPCR assay designed to inform the testing options for couples whose offspring are at risk of suffering from cystic fibrosis via compound heterozygosity. By detecting the presence or absence of the paternal mutation in the cffDNA, it is possible to predict whether the fetus will be an unaffected carrier (absence or whether further invasive testing is indicated (presence.We selected a family in which the parents were known to carry different mutated CFTR alleles as our test system. NIPD was performed for three of their pregnancies during the first trimester (at around 11-12 weeks of gestation. Taqman probes were designed against an amplicon in exon 11 of the CFTR gene, to quantify the proportion of mutant (ΔF508-MUT; FAM and normal (ΔF508-NOR; VIC alleles at position c.1521_1523 of the CFTR gene.The assay correctly and unambiguously recognized the ΔF508-MUT CFTR allele in the cffDNA of all three proband fetuses and none of the six unaffected control fetuses. In conclusion, the Bio-Rad QX100 was found to be a cost-effective and technically undemanding platform for designing bespoke NIPD assays.

  3. A Non-Invasive Droplet Digital PCR (ddPCR) Assay to Detect Paternal CFTR Mutations in the Cell-Free Fetal DNA (cffDNA) of Three Pregnancies at Risk of Cystic Fibrosis via Compound Heterozygosity

    Science.gov (United States)

    Debrand, Emmanuel; Lykoudi, Alexandra; Bradshaw, Elizabeth; Allen, Stephanie K.

    2015-01-01

    Introduction Non-invasive prenatal diagnosis (NIPD) makes use of cell-free fetal DNA (cffDNA) in the mother’s bloodstream as an alternative to invasive sampling methods such as amniocentesis or CVS, which carry a 0.5–1% risk of fetal loss. We describe a droplet digital PCR (ddPCR) assay designed to inform the testing options for couples whose offspring are at risk of suffering from cystic fibrosis via compound heterozygosity. By detecting the presence or absence of the paternal mutation in the cffDNA, it is possible to predict whether the fetus will be an unaffected carrier (absence) or whether further invasive testing is indicated (presence). Methods We selected a family in which the parents were known to carry different mutated CFTR alleles as our test system. NIPD was performed for three of their pregnancies during the first trimester (at around 11–12 weeks of gestation). Taqman probes were designed against an amplicon in exon 11 of the CFTR gene, to quantify the proportion of mutant (ΔF508-MUT; FAM) and normal (ΔF508-NOR; VIC) alleles at position c.1521_1523 of the CFTR gene. Discussion The assay correctly and unambiguously recognized the ΔF508-MUT CFTR allele in the cffDNA of all three proband fetuses and none of the six unaffected control fetuses. In conclusion, the Bio-Rad QX100 was found to be a cost-effective and technically undemanding platform for designing bespoke NIPD assays. PMID:26561302

  4. High levels of fetal DNA are associated with increased risk of spontaneous preterm delivery

    DEFF Research Database (Denmark)

    Jakobsen, Tanja R; Clausen, Frederik B; Rode, Line;

    2012-01-01

    To assess whether spontaneous preterm delivery can be predicted from the amount of cell free fetal DNA (cffDNA) as determined by routine fetal RHD genotyping at 25 weeks' gestation.......To assess whether spontaneous preterm delivery can be predicted from the amount of cell free fetal DNA (cffDNA) as determined by routine fetal RHD genotyping at 25 weeks' gestation....

  5. False Negative Cell-Free DNA Screening Result in a Newborn with Trisomy 13

    OpenAIRE

    Yang Cao; Nicole L. Hoppman; Sarah E Kerr; Sattler, Christopher A.; Borowski, Kristi S.; Wick, Myra J.; Edward Highsmith, W.; Umut Aypar

    2016-01-01

    Background. Noninvasive prenatal screening (NIPS) is revolutionizing prenatal screening as a result of its increased sensitivity, specificity. NIPS analyzes cell-free fetal DNA (cffDNA) circulating in maternal plasma to detect fetal chromosome abnormalities. However, cffDNA originates from apoptotic placental trophoblast; therefore cffDNA is not always representative of the fetus. Although the published data for NIPS testing states that the current technique ensures high sensitivity and speci...

  6. Immune re-activation by cell-free fetal DNA in healthy pregnancies re-purposed to target tumors: novel check-point inhibition in cancer therapeutics

    Directory of Open Access Journals (Sweden)

    Elizabeth Ann Lieser Enninga

    2015-08-01

    Full Text Available The role of the immune system in cancer progression has become increasingly evident over the past decade. Chronic inflammation in the promotion of tumorigenesis is well established, and cancer-associated tolerance/immune evasion has long been appreciated. Recent developments of immunotherapies targeting cancer-associated inflammation and immune tolerance such as cancer vaccines, cell therapies, neutralizing antibodies, and immune checkpoint inhibitors, have shown promising clinical results. However, despite significant therapeutic advances, most patients diagnosed with metastatic cancer still succumb to their malignancy. Treatments are often toxic, and the financial burden of novel therapies is significant. Thus, new methods for utilizing similar biological systems to compare complex biological processes can give us new hypotheses for combating cancer. One such approach is comparing trophoblastic growth and regulation to tumor invasion and immune escape. Novel concepts regarding immune activation in pregnancy, especially reactivation of the immune system at labor through toll like receptor engagement by fetal derived DNA, may be applicable to cancer immunotherapy. This review summarizes mechanisms of inflammation in cancer, current immunotherapies used in the clinic, and suggestions for looking beyond oncology for novel methods to reverse cancer-associated tolerance and immunologic exhaustion utilizing mechanisms encountered in normal human pregnancy.

  7. Immune Reactivation by Cell-Free Fetal DNA in Healthy Pregnancies Re-Purposed to Target Tumors: Novel Checkpoint Inhibition in Cancer Therapeutics

    Science.gov (United States)

    Enninga, Elizabeth Ann L.; Nevala, Wendy K.; Holtan, Shernan G.; Markovic, Svetomir N.

    2015-01-01

    The role of the immune system in cancer progression has become increasingly evident over the past decade. Chronic inflammation in the promotion of tumorigenesis is well established, and cancer-associated tolerance/immune evasion has long been appreciated. Recent developments of immunotherapies targeting cancer-associated inflammation and immune tolerance, such as cancer vaccines, cell therapies, neutralizing antibodies, and immune checkpoint inhibitors, have shown promising clinical results. However, despite significant therapeutic advances, most patients diagnosed with metastatic cancer still succumb to their malignancy. Treatments are often toxic, and the financial burden of novel therapies is significant. Thus, new methods for utilizing similar biological systems to compare complex biological processes can give us new hypotheses for combating cancer. One such approach is comparing trophoblastic growth and regulation to tumor invasion and immune escape. Novel concepts regarding immune activation in pregnancy, especially reactivation of the immune system at labor through toll like receptor engagement by fetal derived DNA, may be applicable to cancer immunotherapy. This review summarizes mechanisms of inflammation in cancer, current immunotherapies used in the clinic, and suggestions for looking beyond oncology for novel methods to reverse cancer-associated tolerance and immunologic exhaustion utilizing mechanisms encountered in normal human pregnancy. PMID:26379664

  8. Research advance in noninvasive prenatal testing based on cell-free fetal DNA%基于胎儿游离DNA的无创产前检测的研究进展

    Institute of Scientific and Technical Information of China (English)

    张展; 赵小辰

    2016-01-01

    母血血浆中胎儿游离DNA( cffDNA)的发现为无创产前检测提供了新思路。虽然目前已经发现多种胎儿DNA标志物,但是如何准确地从母血血浆总游离DNA中区分出cffDNA对我们来说仍然是个难题。目前,基于cffDNA的无创产前检测已被用于多种疾病的检测和研究,随着技术的不断进步和发展,它将会有更广阔的应用前景。本文将从cffDNA的生物学特征、标志物,cffDNA的无创产前检测的临床应用及其现阶段存在的问题和发展前景等方面进行阐述。(中华检验医学杂志,2016,39:307-310)%The discovery of cell-free fetal DNA ( cffDNA) in maternal plasma provides a new idea for noninvasive prenatal testing( NIPT).Though some studies to date have shown several fetal DNA markers, how to accurately distinguish cffDNA from the pool of maternal plasma free DNA is still a challenge.So far, NIPT based on cffDNA has been used for detection and study of a variety of diseases, along with the advance and development of technology, it will have a more broad application prospects.This article will make a review for the research status from the biological characteristics and the markers of cffDNA, the clinical applications and the existing issues and development prospects of NIPT based on cffDNA.

  9. Cell-free DNA: Preanalytical variables.

    Science.gov (United States)

    Bronkhorst, Abel Jacobus; Aucamp, Janine; Pretorius, Piet J

    2015-10-23

    Since the discovery of cell-free DNA (cfDNA) in human blood, most studies have focused on diagnostic and prognostic uses of these markers for solid tumors. Except for some prenatal tests and BEAMing, cfDNA analysis has not yet been translated to clinical practice and routine application appears distant. This can be attributed to overlapping factors: (i) a lack of knowledge regarding the origin and function of cfDNA, (ii) insufficient molecular characterization, and (iii) the absence of an analytical consensus. In this review, we address the latter determinant and focus specifically on quantitative analysis of cfDNA. While the literature reports limited value for a single quantitative assessment, cfDNA kinetic assessment will be an essential component to qualitative characterization. In order to establish quantitative analysis for accurate kinetic assessments, process optimization and standardization are crucial. This report elucidates the most confounding variables of each preanalytic step that must be considered for optimal analysis. PMID:26341895

  10. Vanished Twins and Misdiagnosed Sex: A Case Report with Implications in Prenatal Counseling Using Noninvasive Cell-Free DNA Screening.

    Science.gov (United States)

    Kelley, James F; Henning, George; Ambrose, Anthony; Adelman, Alan

    2016-01-01

    Cell-free DNA testing is a recently introduced method for screening pregnant women for fetal trisomy, which is associated with some common significant genetic diseases, as well as the sex of the fetus. The case described here demonstrates the connection between the ultrasound "vanishing twin" phenomenon and the misdiagnosis of prenatal sex using cell-free DNA testing. PMID:27170800

  11. 孕妇外周血中无细胞胎儿DNA的研究进展及应用%Progress for cell free fetal DNA from maternal peripheral blood and its applications

    Institute of Scientific and Technical Information of China (English)

    杨维婵; 傅晓冬; 傅俊江

    2015-01-01

    Cell free fetal DNA (cffDNA) in maternal peripheral blood is an important material of fetus for detection in non-invasive prenatal diagnosis (NIPD).Most DNAs in maternal blood is maternal origin,the proportion of cffDNA is just 3 % ~ 6 %.Therefore,successful isolation of cffDNA from maternal peripheral blood plays an important role in the subsequent NIPT.In this paper we will review the discovery,origin,structure and stability of cffDNA laboratory method of isolation from maternal blood,and the its application in NIPT,and emphasize on the recently research progress of technology systematically.The purpose is to explore the laboratory method for cffDNA isolation with high efficiency,to provide a high concentration of cffDNA for NIPT and increase the accuracy rate and success rate in NIPD.%孕妇外周血中无细胞胎儿DNA(cffDNA)是无创性产前诊断中重要的胎儿物质的检测来源.由于孕妇血中大部分是母体DNA,而游离胎儿DNA的量非常少,仅占3%~6%.因此从孕妇血中成功分离cffDNA,对后续的无创性产前诊断有着十分重要的意义.本文分别从孕妇外周血中cffDNA的发现来源,cffDNA的结构与稳定性,分离孕妇外周血中的cffDNA的实验方法,及在无创性产前诊断中的应用等方面进行介绍,并着重对该技术近年来的研究进展作一综述.旨在探寻较高效率分离孕妇外周血中cffDNA的实验方法,为无创性产前诊断提供较高浓度的检测物质,提高其准确率及成功率.

  12. Značaj ukupne i fetalne slobodne DNA iz majčine krvi u neinvazivnom otkrivanju fetalnih aneuploidija [The significance of total and fetal cell free DNA from maternal blood in noninvasive detection of fetal aneuploidies

    OpenAIRE

    Bekavac Vlatković, Ivanka

    2015-01-01

    INTRODUCTION: Chromosomal abnormalities account for approximately 15% of the major congenital anomalies before the age 1 year in Europe, and are associated with 25% of perinatal deaths due to congenital anomalies. Maternal serum is routinely analyzed in pregnant women to screen for chromosomal abnormalities such as Down and Edwards syndrome. In addition there are many reports of an association between abnormal levels of individual analytes and poor fetal or placental health. A low pregnanc...

  13. 利用孕妇血浆中的胎儿DNA进行β-地中海贫血的产前诊断%Prenatal diagnosis of β-thalassaemia using cell-free fetal DNA in maternal plasma

    Institute of Scientific and Technical Information of China (English)

    李广华; 荣卡彬; 罗燕飞; 陈冬; 龚彩平; 吴劲; 邸玉玮; 葛艳芬

    2011-01-01

    目的 利用孕妇血浆中游离胎儿DNA(cffDNA)对广东省最常见17种β地中海贫血的突变基因进行扩增,探讨非创伤性产前诊断β-地中海贫血的可行性.方法 ①羊水途径:抽取孕妇羊水共9例,采用反向斑点杂交技术检测中国人群8个常见位点和9个少见位点突变的共17种β地贫基因;②抽取孕妇外周血,柱分离法提取及凝胶回收纯化DNA,设计3对引物,对cffDNA进行二次PCR,反向斑点杂交技术检测β地中海贫血的突变基因.结果 9例孕妇中有5例经羊水检测证实胎儿有父系的β地贫基因,有2例孕妇外周血中检测到胎儿(父系)β地贫基因,与羊水检测相符.结论 利用cffDNA进行β-地中海贫血的检测方法可行,但由于母源性DNA背景的污染,以及胎儿DNA因含量而导致检出率低,希望进一步改进该技术后有望可用于β-地中海贫血的诊断.%Objective To investigate the clinical feasibility of cell-free fetal DNA (cffDNA)-based noninvasive prenatal diagnosis of β-thalassemia. Methods Nine samples of amniotic fluid were obtained to detect the 8 common and 9 relatively rare mutation sites of [3-thalassaemia in Guangdong Province. The maternal blood samples were also collected for extracting and purification of the cffDNA, and a duplex PCR was performed using 3 pairs of primers and the fetal β-globin genotype was analyzed by reverse dot-blot hybridization. Results Among the 9 cases, 5 showed fetal genotypes of β-thalassemia inherited from the father by examination of the amniotic fluid, and 2 fetuses were identified to have β-thalassemia genes inherited from the father determined based on the cffDNA in the maternal blood. Conclusion The cffDNA-based noninvasive prenatal diagnosis is feasible for β-thalassemia, but the contamination of the maternal background DNA results in a low detection rate.

  14. Fetal RHD Genotyping Using Real-Time Polymerase Chain Reaction Analysis of Cell-Free Fetal DNA in Pregnancy of RhD Negative Women in South of Iran

    OpenAIRE

    Leili Moezzi; Zeinab Keshavarz; Reza Ranjbaran; Farzaneh Aboualizadeh; Abbas Behzad-Behbahani; Masooma Abdullahi; Amin Ramezani; Alamtaj Samsami; Sedigheh Sharifzadeh

    2016-01-01

    Background: Maternal-fetal RhD antigen incompatibility causes approximately 50% of clinically significant alloimmunization cases. The routine use of prophylactic anti-D immunoglobulin has dramatically reduced hemolytic disease of the fetus and newborn. Recently, fetal RHD genotyping in RhD negative pregnant women has been suggested for appropriate use of anti-D immunoglobulin antenatal prophylaxis and decrease unnecessary prenatal interventions. Materials and Methods: In thi...

  15. Cell-free fetal DNA in maternal plasma and noninvasive prenatal diagnosis DNA fetal libre en el plasma materno y diagnóstico prenatal no invasivo DNA livre fetal em plasma materno e diagnóstico pré-natal não invasivo

    Directory of Open Access Journals (Sweden)

    Ester Silveira Ramos

    2006-12-01

    Full Text Available The noninvasive nature of the detection of fetal DNA in the maternal circulation represents the greatest advantage over the conventional methods of prenatal diagnosis. The applications of this methodology involve the detection of the fetal sex, and diagnosis, intra-uterine treatment, and evaluation of the prognosis of many diseases. Fetal cells detected in the maternal circulation have also been shown to be implicated in autoimmune diseases and to represent a potential source of stem cells. On the other hand, with the introduction of a technology that detects the fetal sex as early as at 6-8 weeks of gestation, there is the possibility of early abortion based on sex selection for social purposes. This implies an ethical discussion about the question. The introduction of new noninvasive techniques of prenatal diagnosis and the knowledge of the Nursing Team regarding new methodologies can be of great benefit to the mother and her children, and can help the Genetic Counseling of the families.La naturaleza no invasiva de la investigación del DNA fetal en la circulación materna representa una ventaja importante con relación a los métodos convencionales de diagnóstico prenatal. El uso de esta metodología implica la determinación del sexo fetal y el diagnóstico, el tratamiento intra-útero y la evaluación del pronóstico en muchas enfermedades. Las células fetales detectadas en la circulación maternal también pueden ser implicadas en enfermedades autoinmunes y representar una fuente potencial de células madre. Por otra parte, con la introducción de una tecnología que detecte el sexo fetal entre 6-8 semanas de gestación, existe la posibilidad de aborto precoz basada en la selección del sexo para los propósitos sociales. Esto implica una discusión ética previa sobre este problema. La introducción de nuevas técnicas no invasivas de diagnóstico prenatal y el conocimiento del Equipo de Enfermería con respecto a las nuevas metodolog

  16. Prenatal assessment of fetal chromosomal and genetic disorders through maternal plasma DNA analysis.

    Science.gov (United States)

    Liao, Gary J W; Chiu, Rossa W K; Lo, Y M Dennis

    2012-02-01

    The existence of cell free DNA derived from the fetus in the plasma of pregnant women was first demonstrated in 1997. This discovery offered the possibility of non-invasive sampling of fetal genetic material simply through the collection of a maternal blood sample. Such cell free fetal DNA molecules in the maternal circulation have subsequently been shown to originate from the placenta and could be detected from about 7 weeks of gestation. It has been shown that cell free fetal DNA analysis could offer highly accurate assessment of fetal genotype and chromosomal makeup for some applications. Thus, cell free fetal DNA analysis has been incorporated as a part of prenatal screening programs for the prenatal management of sex-linked and sex-associated diseases, rhesus D incompatibility as well as the prenatal detection of Down's syndrome.Cell free fetal DNA analysis may lead to a change in the way prenatal assessments are made. PMID:22198255

  17. Nonhomologous DNA End Joining in Cell-Free Extracts

    Directory of Open Access Journals (Sweden)

    Sheetal Sharma

    2010-01-01

    Full Text Available Among various DNA damages, double-strand breaks (DSBs are considered as most deleterious, as they may lead to chromosomal rearrangements and cancer when unrepaired. Nonhomologous DNA end joining (NHEJ is one of the major DSB repair pathways in higher organisms. A large number of studies on NHEJ are based on in vitro systems using cell-free extracts. In this paper, we summarize the studies on NHEJ performed by various groups in different cell-free repair systems.

  18. False Negative Cell-Free DNA Screening Result in a Newborn with Trisomy 13

    Directory of Open Access Journals (Sweden)

    Yang Cao

    2016-01-01

    Full Text Available Background. Noninvasive prenatal screening (NIPS is revolutionizing prenatal screening as a result of its increased sensitivity, specificity. NIPS analyzes cell-free fetal DNA (cffDNA circulating in maternal plasma to detect fetal chromosome abnormalities. However, cffDNA originates from apoptotic placental trophoblast; therefore cffDNA is not always representative of the fetus. Although the published data for NIPS testing states that the current technique ensures high sensitivity and specificity for aneuploidy detection, false positives are possible due to isolated placental mosaicism, vanishing twin or cotwin demise, and maternal chromosome abnormalities or malignancy. Results. We report a case of false negative cell-free DNA (cfDNA screening due to fetoplacental mosaicism. An infant male with negative cfDNA screening result was born with multiple congenital abnormalities. Postnatal chromosome and FISH studies on a blood specimen revealed trisomy 13 in 20/20 metaphases and 100% interphase nuclei, respectively. FISH analysis on tissues collected after delivery revealed extraembryonic mosaicism. Conclusions. Extraembryonic tissue mosaicism is likely responsible for the false negative cfDNA screening result. This case illustrates that a negative result does not rule out the possibility of a fetus affected with a trisomy, as cffDNA is derived from the placenta and therefore may not accurately represent the fetal genetic information.

  19. False Negative Cell-Free DNA Screening Result in a Newborn with Trisomy 13

    Science.gov (United States)

    Cao, Yang; Hoppman, Nicole L.; Kerr, Sarah E.; Sattler, Christopher A.; Borowski, Kristi S.; Wick, Myra J.; Highsmith, W. Edward; Aypar, Umut

    2016-01-01

    Background. Noninvasive prenatal screening (NIPS) is revolutionizing prenatal screening as a result of its increased sensitivity, specificity. NIPS analyzes cell-free fetal DNA (cffDNA) circulating in maternal plasma to detect fetal chromosome abnormalities. However, cffDNA originates from apoptotic placental trophoblast; therefore cffDNA is not always representative of the fetus. Although the published data for NIPS testing states that the current technique ensures high sensitivity and specificity for aneuploidy detection, false positives are possible due to isolated placental mosaicism, vanishing twin or cotwin demise, and maternal chromosome abnormalities or malignancy. Results. We report a case of false negative cell-free DNA (cfDNA) screening due to fetoplacental mosaicism. An infant male with negative cfDNA screening result was born with multiple congenital abnormalities. Postnatal chromosome and FISH studies on a blood specimen revealed trisomy 13 in 20/20 metaphases and 100% interphase nuclei, respectively. FISH analysis on tissues collected after delivery revealed extraembryonic mosaicism. Conclusions. Extraembryonic tissue mosaicism is likely responsible for the false negative cfDNA screening result. This case illustrates that a negative result does not rule out the possibility of a fetus affected with a trisomy, as cffDNA is derived from the placenta and therefore may not accurately represent the fetal genetic information. PMID:26998368

  20. An Economic Analysis of Cell-Free DNA Non-Invasive Prenatal Testing in the US General Pregnancy Population

    OpenAIRE

    Benn, Peter; Curnow, Kirsten J.; Chapman, Steven; Michalopoulos, Steven N.; Hornberger, John; Rabinowitz, Matthew

    2015-01-01

    Objective Analyze the economic value of replacing conventional fetal aneuploidy screening approaches with non-invasive prenatal testing (NIPT) in the general pregnancy population. Methods Using decision-analysis modeling, we compared conventional screening to NIPT with cell-free DNA (cfDNA) analysis in the annual US pregnancy population. Sensitivity and specificity for fetal aneuploidies, trisomy 21, trisomy 18, trisomy 13, and monosomy X, were estimated using published data and modeling of b...

  1. Quantitative analysis of cell-free DNA in ovarian cancer

    OpenAIRE

    Shao, Xuefeng; He, Yan; Ji, Min; Chen, Xiaofang; Qi, Jing; SHI, Wei; HAO, TIANBO; JU, SHAOQING

    2015-01-01

    The aim of the present study was to investigate the association between cell-free DNA (cf-DNA) levels and clinicopathological characteristics of patients with ovarian cancer using a branched DNA (bDNA) technique, and to determine the value of quantitative cf-DNA detection in assisting with the diagnosis of ovarian cancer. Serum specimens were collected from 36 patients with ovarian cancer on days 1, 3 and 7 following surgery, and additional serum samples were also collected from 22 benign ova...

  2. Circulating Cell Free DNA in the Diagnosis of Trophoblastic Tumors

    OpenAIRE

    Openshaw, Mark R.; Harvey, Richard A.; Sebire, Neil J; Baljeet Kaur; Naveed Sarwar; Michael J Seckl; Fisher, Rosemary A.

    2015-01-01

    Gestational trophoblastic neoplasia (GTN) represents a group of diseases characterized by production of human chorionic gonadotropin (hCG). Since non-gestational tumors may occasionally secrete hCG, histopathological diagnosis is important for appropriate clinical management. However, a histopathological diagnosis is not always available. We therefore investigated the feasibility of extracting cell free DNA (cfDNA) from the plasma of women with GTN for use as a “liquid biopsy” in patients wit...

  3. Decreased serum cell-free DNA levels in rheumatoid arthritis

    OpenAIRE

    Dunaeva, Marina; Buddingh’, Bastiaan C.; René E M Toes; Luime, Jolanda J.; Lubberts, Erik; Pruijn, Ger J. M.

    2015-01-01

    Purpose Recent studies have demonstrated that serum/plasma DNA and RNA molecules in addition to proteins can serve as biomarkers. Elevated levels of these nucleic acids have been found not only in acute, but also in chronic conditions, including autoimmune diseases. The aim of this study was to assess cell-free DNA (cfDNA) levels in sera of rheumatoid arthritis (RA) patients compared to controls. Methods cfDNA was extracted from sera of patients with early and established RA, relapsing-remitt...

  4. High-resolution characterization of sequence signatures due to non-random cleavage of cell-free DNA

    OpenAIRE

    Chandrananda, Dineika; Thorne, Natalie P.; Bahlo, Melanie

    2015-01-01

    Background High-throughput sequencing of cell-free DNA fragments found in human plasma has been used to non-invasively detect fetal aneuploidy, monitor organ transplants and investigate tumor DNA. However, many biological properties of this extracellular genetic material remain unknown. Research that further characterizes circulating DNA could substantially increase its diagnostic value by allowing the application of more sophisticated bioinformatics tools that lead to an improved signal to n...

  5. Cell-free DNA testing: an aid to prenatal sonographic diagnosis.

    Science.gov (United States)

    Chitty, Lyn S

    2014-04-01

    Sonographic diagnosis of fetal abnormalities is based on the recognition of sonographic patterns associated with structural abnormalities. Although diagnosis in some situations, such as neural tube defects, gastroschisis, and omphalocoele, can be straightforward, in many situations, the constellation of fetal abnormalities suggest an underlying chromosomal or genetic cause. In these situations, invasive testing is needed to provide the information required to make a definitive diagnosis, and thus accurately counsel parents. Since the identification of cell-free fetal DNA in maternal plasma, the potential for non-invasive prenatal diagnosis is increasingly becoming possible. In this chapter, the current role and future potential of non-invasive prenatal diagnosis, combined with new molecular techniques as an aid to sonographic diagnosis, will be discussed. PMID:24594366

  6. Quantification of cell-free DNA in normal and complicated pregnancies: overcoming biological and technical issues.

    Directory of Open Access Journals (Sweden)

    Irina Manokhina

    Full Text Available The characterization of cell-free DNA (cfDNA originating from placental trophoblast in maternal plasma provides a powerful tool for non-invasive diagnosis of fetal and obstetrical complications. Due to its placental origin, the specific epigenetic features of this DNA (commonly known as cell-free fetal DNA can be utilized in creating universal 'fetal' markers in maternal plasma, thus overcoming the limitations of gender- or rhesus-specific ones. The goal of this study was to compare the performance of relevant approaches and assays evaluating the amount of cfDNA in maternal plasma throughout gestation (7.2-39.5 weeks. Two fetal- or placental-specific duplex assays (RPP30/SRY and RASSF1A/β-Actin were applied using two technologies, real-time quantitative PCR (qPCR and droplet digital PCR (ddPCR. Both methods revealed similar performance parameters within the studied dynamic range. Data obtained using qPCR and ddPCR for these assays were positively correlated (total cfDNA (RPP30: R = 0.57, p = 0.001/placental cfDNA (SRY: R = 0.85, p<0.0001; placental cfDNA (RASSF1A: R = 0.75, p<0.0001. There was a significant correlation in SRY and RASSF1A results measured with qPCR (R = 0.68, p = 0.013 and ddPCR (R = 0.56, p = 0.039. Different approaches also gave comparable results with regard to the correlation of the placental cfDNA concentration with gestational age and pathological outcome. We conclude that ddPCR is a practical approach, adaptable to existing qPCR assays and well suited for analysis of cell-free DNA in plasma. However, it may need further optimization to surpass the performance of qPCR.

  7. Controls to validate plasma samples for cell free DNA quantification

    DEFF Research Database (Denmark)

    Pallisgaard, Niels; Spindler, Karen-Lise Garm; Andersen, Rikke Fredslund;

    2015-01-01

    Recent research has focused on the utility of cell free DNA (cfDNA) in serum and plasma for clinical application, especially in oncology. The literature holds promise of cfDNA as a valuable tumour marker to be used for treatment selection, monitoring and follow-up. The results, however, are...... diverging due to methodological differences with lack of standardisation and definition of sensitivity. The new biological information has not yet come into routine use. The present study presents external standardisation by spiking with non-human DNA fragments to control for loss of DNA during sample...... preparation and measurement. It also suggests a method to control for admixture of DNA from normal lymphocytes by utilizing the unique immunoglobulin gene rearrangement in the B-cells. The results show that this approach improves the quality of the analysis and lowers the risk of falsely increased values. In...

  8. Circulating Cell Free DNA in the Diagnosis of Trophoblastic Tumors

    Directory of Open Access Journals (Sweden)

    Mark R. Openshaw

    2016-02-01

    Full Text Available Gestational trophoblastic neoplasia (GTN represents a group of diseases characterized by production of human chorionic gonadotropin (hCG. Since non-gestational tumors may occasionally secrete hCG, histopathological diagnosis is important for appropriate clinical management. However, a histopathological diagnosis is not always available. We therefore investigated the feasibility of extracting cell free DNA (cfDNA from the plasma of women with GTN for use as a “liquid biopsy” in patients without histopathological diagnosis. cfDNA was prepared from the plasma of 20 women with a diagnosis of GTN and five with hCG-secreting tumors of unknown origin. Genotyping of cfDNA from the patient, genomic DNA from her and her partner and DNA from the tumor tissue identified circulating tumor DNA (ctDNA (from 9% to 53% of total cfDNA in 12 of 20 patients with GTN. In one case without a tissue diagnosis, ctDNA enabled a diagnosis of GTN originating in a non-molar conception and in another a diagnosis of non-gestational tumor, based on the high degree of allelic instability and loss of heterozygosity in the ctDNA. In summary ctDNA can be detected in the plasma of women with GTN and can facilitate the diagnosis of both gestational and non-gestational trophoblastic tumors in cases without histopathological diagnosis.

  9. Strategies for Implementing Cell-Free DNA Testing.

    Science.gov (United States)

    Cuckle, Howard

    2016-06-01

    Maternal plasma cell-free (cf) DNA testing has higher discriminatory power for aneuploidy than any conventional multi-marker screening test. Several strategies have been suggested for introducing it into clinical practice. Secondary cfDNA, restricted only to women with positive conventional screening test, is generally cost saving and minimizes the need for invasive prenatal diagnosis but leads to a small loss in detection. Primary cfDNA, replacing conventional screening or retaining the nuchal translucency scan, is not currently cost-effective for third-party payers. Contingent cfDNA, testing about 20% of women with the highest risks based on a conventional test, is the preferred approach. PMID:27235907

  10. The Long and Short of Circulating Cell-Free DNA and the Ins and Outs of Molecular Diagnostics.

    Science.gov (United States)

    Jiang, Peiyong; Lo, Y M Dennis

    2016-06-01

    The discovery of cell-free tumor and fetal DNA molecules in the plasma of cancer patients and pregnant women, respectively, has opened up exciting opportunities in molecular diagnosis. The understanding of the biological properties of circulating cell-free DNA (cfDNA) molecules would be essential for us to make the best use of such molecules in different clinical settings. In this review we start by exploring the technologies that have been used for analyzing the size profiles of cfDNA in plasma. We then review the size profiles of cfDNA in different clinical scenarios, including cancer, pregnancy, transplantation, and autoimmune diseases. Finally, we discuss the potential diagnostic applications of plasma DNA size profiling. PMID:27129983

  11. Detection of cell-free fetal DNA in maternal plasma of the women with pregnancy, who have high-risk of carrying a Down's fetus in Down's syndrome screening%唐氏综合征高危孕妇血浆游离胎儿DNA的检测

    Institute of Scientific and Technical Information of China (English)

    王靖; 陈汉平

    2011-01-01

    Objective: To investigate the value of Realtime PCR ( RQ-PCR) for detection of fetal DNA in maternal plasma to screen the high-risk women in Down's syndrome screening. Methods: We seclected 42 cases of second trimester pregnant women, 22 cases have high-risk of earring a Down's fetus and 20 cases normal. Using RQ-PCR to detect SRY and GAPDH genes in maternal plasma, the varince between the two groups was analysizd by 2△△C1 method. Results; SRY was detected in all 22 women with male fetus, but SRY was detected in 2 cases of 20 women with femal fetus. The amount of cell-free fetal DNA was significantly higher in high-risk pregnant women than normal (P = 0. 006, < 0.05 ) , the ratio was 2. 79. Conclusion: The quantitative analysis of cell-free fetal DNA in maternal plasma is of great value in the Down's syndrome screening.%目的 探讨实时荧光定量PCR(RQ-PCR)检测孕妇血浆游离胎儿DNA在筛查唐氏综合征高危孕妇中的应用.方法 采用RQ-PCR检测22例唐氏综合征高危孕妇及20例低危孕妇血浆中GAPDH及SRY水平,2-△△Ct法分析两组间的差异.结果 22例孕男胎均检出SRY基因,20例孕女胎中出现2例假阳性,高危组游离胎儿DNA水平明显高于低危组(P=0.006,<0.05),比值为2.79.结论 孕妇血浆游离胎儿DNA的定量检测在唐氏综合征筛查中有重要价值.

  12. Controls to validate plasma samples for cell free DNA quantification.

    Science.gov (United States)

    Pallisgaard, Niels; Spindler, Karen-Lise Garm; Andersen, Rikke Fredslund; Brandslund, Ivan; Jakobsen, Anders

    2015-06-15

    Recent research has focused on the utility of cell free DNA (cfDNA) in serum and plasma for clinical application, especially in oncology. The literature holds promise of cfDNA as a valuable tumour marker to be used for treatment selection, monitoring and follow-up. The results, however, are diverging due to methodological differences with lack of standardisation and definition of sensitivity. The new biological information has not yet come into routine use. The present study presents external standardisation by spiking with non-human DNA fragments to control for loss of DNA during sample preparation and measurement. It also suggests a method to control for admixture of DNA from normal lymphocytes by utilizing the unique immunoglobulin gene rearrangement in the B-cells. The results show that this approach improves the quality of the analysis and lowers the risk of falsely increased values. In conclusion we suggest a new method to improve the accuracy of cfDNA measurements easily incorporated in the current technology. PMID:25896958

  13. Cell-free circulating tumor DNA in cancer.

    Science.gov (United States)

    Qin, Zhen; Ljubimov, Vladimir A; Zhou, Cuiqi; Tong, Yunguang; Liang, Jimin

    2016-01-01

    Cancer is a common cause of death worldwide. Despite significant advances in cancer treatments, the morbidity and mortality are still enormous. Tumor heterogeneity, especially intratumoral heterogeneity, is a significant reason underlying difficulties in tumor treatment and failure of a number of current therapeutic modalities, even of molecularly targeted therapies. The development of a virtually noninvasive "liquid biopsy" from the blood has been attempted to characterize tumor heterogeneity. This review focuses on cell-free circulating tumor DNA (ctDNA) in the bloodstream as a versatile biomarker. ctDNA analysis is an evolving field with many new methods being developed and optimized to be able to successfully extract and analyze ctDNA, which has vast clinical applications. ctDNA has the potential to accurately genotype the tumor and identify personalized genetic and epigenetic alterations of the entire tumor. In addition, ctDNA has the potential to accurately monitor tumor burden and treatment response, while also being able to monitor minimal residual disease, reducing the need for harmful adjuvant chemotherapy and allowing more rapid detection of relapse. There are still many challenges that need to be overcome prior to this biomarker getting wide adoption in the clinical world, including optimization, standardization, and large multicenter trials. PMID:27056366

  14. Quantitative analysis of cell-free DNA in ovarian cancer

    Science.gov (United States)

    SHAO, XUEFENG; He, YAN; JI, MIN; CHEN, XIAOFANG; QI, JING; SHI, WEI; HAO, TIANBO; JU, SHAOQING

    2015-01-01

    The aim of the present study was to investigate the association between cell-free DNA (cf-DNA) levels and clinicopathological characteristics of patients with ovarian cancer using a branched DNA (bDNA) technique, and to determine the value of quantitative cf-DNA detection in assisting with the diagnosis of ovarian cancer. Serum specimens were collected from 36 patients with ovarian cancer on days 1, 3 and 7 following surgery, and additional serum samples were also collected from 22 benign ovarian tumor cases, and 19 healthy, non-cancerous ovaries. bDNA techniques were used to detect serum cf-DNA concentrations. All data were analyzed using SPSS version 18.0. The cf-DNA levels were significantly increased in the ovarian cancer group compared with those of the benign ovarian tumor group and healthy ovarian group (P<0.01). Furthermore, cf-DNA levels were significantly increased in stage III and IV ovarian cancer compared with those of stages I and II (P<0.01). In addition, cf-DNA levels were significantly increased on the first day post-surgery (P<0.01), and subsequently demonstrated a gradual decrease. In the ovarian cancer group, the area under the receiver operating characteristic curve of cf-DNA and the sensitivity were 0.917 and 88.9%, respectively, which was higher than those of cancer antigen 125 (0.724, 75%) and human epididymis protein 4 (0.743, 80.6%). There was a correlation between the levels of serum cf-DNA and the occurrence and development of ovarian cancer in the patients evaluated. bDNA techniques possessed higher sensitivity and specificity than other methods for the detection of serum cf-DNA in patients exhibiting ovarian cancer, and bDNA techniques are more useful for detecting cf-DNA than other factors. Thus, the present study demonstrated the potential value for the use of bDNA as an adjuvant diagnostic method for ovarian cancer. PMID:26788153

  15. Advances on circulating fetal DNA in maternal plasma

    Institute of Scientific and Technical Information of China (English)

    FU Xian-hu; CHEN Han-ping

    2007-01-01

    @@ The discovery of cell-free fetal DNA in maternal plasma in 1997 has opened up new possibilities for noninvasive diagnosis.1 By RT-PCR, circulating fetal DNA can be detected in the plasma of pregnant women,even in the first trimester of pregnancy,2,3 and thus can be used for noninvasive prenatal diagnosis of sex-linked disorders,4-6 the RhD status of fetuses,7 and single gene disorders such as beta-thalassaemia,8,9 congenital adrenal hyperplasia,10 and achondroplasia.11 In addition,quantitative aberrations of circulating fetal DNA may indicate various pregnancy-associated disorders,including1 Preeclampsia,12-14 preterm labor15,16 and fetal trisomy 21.17

  16. Multiparametric analysis of cell-free DNA in melanoma patients.

    Directory of Open Access Journals (Sweden)

    Francesca Salvianti

    Full Text Available Cell-free DNA in blood (cfDNA represents a promising biomarker for cancer diagnosis. Total cfDNA concentration showed a scarce discriminatory power between patients and controls. A higher specificity in cancer diagnosis can be achieved by detecting tumor specific alterations in cfDNA, such as DNA integrity, genetic and epigenetic modifications.The aim of the present study was to identify a sequential multi-marker panel in cfDNA able to increase the predictive capability in the diagnosis of cutaneous melanoma in comparison with each single marker alone. To this purpose, we tested total cfDNA concentration, cfDNA integrity, BRAF(V600E mutation and RASSF1A promoter methylation associated to cfDNA in a series of 76 melanoma patients and 63 healthy controls. The chosen biomarkers were assayed in cfDNA samples by qPCR. Comparison of biomarkers distribution in cases and controls was performed by a logistic regression model in both univariate and multivariate analysis. The predictive capability of each logistic model was investigated by means of the area under the ROC curve (AUC. To aid the reader to interpret the value of the AUC, values between 0.6 and 0.7, between 0.71 and 0.8 and greater than 0.8 were considered as indicating a weak predictive, satisfactory and good predictive capacity, respectively. The AUC value for each biomarker (univariate logistic model was weak/satisfactory ranging between 0.64 (BRAF(V600E to 0.85 (total cfDNA. A good overall predictive capability for the final logistic model was found with an AUC of 0.95. The highest predictive capability was given by total cfDNA (AUC:0.86 followed by integrity index 180/67 (AUC:0.90 and methylated RASSF1A (AUC:0.89.An approach based on the simultaneous determination of three biomarkers (total cfDNA, integrity index 180/67 and methylated RASSF1A could improve the diagnostic performance in melanoma.

  17. Detection of Y STR markers of male fetal dna in maternal circulation

    OpenAIRE

    Nair Seema; Peter Sam; Pillay V; Remya U; Krishnaprasad R; Rajammal B

    2007-01-01

    Background: Circulating fetal cells and cell free DNA in the maternal blood has been shown to help in prenatal diagnosis of genetic disorders without relying on invasive procedures leading to significant risk of pregnancy loss. Aim: The current study was undertaken to detect the male fetal population using Y STR markers DYS 19, DYS 385 and DYS 392 and also to study the extent of persistence of fetal DNA in the mother following delivery. Materials and Methods: Blinded study was conducted ...

  18. Detection of Y STR markers of male fetal dna in maternal circulation

    Directory of Open Access Journals (Sweden)

    Nair Seema

    2007-01-01

    Full Text Available Background: Circulating fetal cells and cell free DNA in the maternal blood has been shown to help in prenatal diagnosis of genetic disorders without relying on invasive procedures leading to significant risk of pregnancy loss. Aim: The current study was undertaken to detect the male fetal population using Y STR markers DYS 19, DYS 385 and DYS 392 and also to study the extent of persistence of fetal DNA in the mother following delivery. Materials and Methods: Blinded study was conducted on 50 mothers delivering male and female babies. Cellular and cell free DNA was extracted from maternal and fetal cord blood and amplified for Y STR markers by PCR. Results: The amplification sensitivity of Y specific STR, DYS19 was 100% (22/22 in the male fetal DNA samples. The incidence of other STRs, i.e., DYS385 and DYS392 were 91% (20/22 each. Analysis of results revealed that thirteen of the twenty six women had detectable male fetal DNA at the time of delivery. However fetal DNA was not detectable twenty four hours after delivery. Conclusion: Preliminary results show that the separation of fetal cell-free DNA in the maternal circulation is a good low-cost approach for the future development of novel strategies to provide non-invasive techniques for early prenatal diagnosis.

  19. Quantification of circulating cell-free DNA in the plasma of cancer patients during radiation therapy

    International Nuclear Information System (INIS)

    Cell-free plasma DNA is elevated in cancer patients and decreases in response to effective treatments. Consequently, these nucleic acids have potential as new tumor markers. In our current study, we investigated whether the plasma DNA concentrations in patients with cancer are altered during the course of radiation therapy. To first determine the origin of cell-free plasma DNA, plasma samples from mice bearing transplanted human tumors were analyzed for human-specific and mouse-specific cell-free DNA. Human-specific DNA was detectable only in plasma from tumor-bearing mice. However, mouse-specific plasma DNA was significantly higher in tumor-bearing mice than in normal mice, suggesting that cell-free plasma DNA originated from both tumor and normal cells. We measured the total cell-free plasma DNA levels by quantitative polymerase chain reaction in 15 cancer patients undergoing radiation therapy and compared these values with healthy control subjects. The cancer patients showed higher pretreatment plasma DNA concentrations than the healthy controls. Eleven of these patients showed a transient increase of up to eightfold in their cell-free plasma DNA concentrations during the first or second week of radiation therapy, followed by decreasing concentrations toward the end of treatment. In two other cancer patients, the cell-free plasma DNA concentrations only decreased over the course of the treatment. The total cell-free plasma DNA levels in cancer patients thus show dynamic changes associated with the progression of radiation therapy. Additional prospective studies will be required to elucidate the potential clinical utility and biological implications of dynamic changes in cell-free plasma DNA during radiation therapy. (author)

  20. Improved recovery of bisulphite-treated cell-free DNA in plasma

    DEFF Research Database (Denmark)

    Pedersen, Inge Søkilde; Krarup, H.B.; Thorlacius-Ussing, O.;

    Detection of cell-free methylated DNA in plasma is a promising tool for tumour diagnosis and monitoring. Due to the very low amount of cell-free DNA in plasma, sensitivity of the detection methods are of utmost importance. The vast majority of currently available methods for analysing DNA...... of PCR amplifying methylated and umethylated MEST. This procedure allows low levels of DNA to be easily and reliably analysed, a prerequisite for the clinical usefulness of cell-free methylated DNA detection in plasma....... methylation are based on bisulphite-mediated deamination of cytosine. However, the recovery of bisulphite-converted DNA is very poor. Here we introduce an alternative method for the crucial steps of bisulphite removal and desulfonation, improving recovery, especially for specimens with low levels of DNA. The...

  1. Chimeric External Control to Quantify Cell Free DNA in Plasma Samples by Real Time PCR

    OpenAIRE

    Eini, Maryam; Behzad-Behbahani, Abbas; Takhshid, Mohammad Ali; Ramezani, Amin; Rafiei Dehbidi, Gholam Reza; Okhovat, Mohammad Ali; Farhadi, Ali; Alavi, Parniyan

    2016-01-01

    Background: DNA isolation procedure can significantly influence the quantification of DNA by real time PCR specially when cell free DNA (cfDNA) is the subject. To assess the extraction efficiency, linearity of the extraction yield, presence of co-purified inhibitors and to avoid problems with fragment size relevant to cfDNA, development of appropriate External DNA Control (EDC) is challenging. Using non-human chimeric nucleotide sequences, an EDC was developed for standardization of qPCR for ...

  2. Plasma HER2 amplification in cell-free DNA during neoadjuvant chemotherapy in breast cancer

    DEFF Research Database (Denmark)

    Bechmann, Troels; Andersen, Rikke Fredslund; Pallisgaard, Niels;

    2013-01-01

    Measurement of human epidermal growth factor receptor 2 (HER2) gene amplification in cell-free DNA (cfDNA) is an evolving technique in breast cancer, enabling liquid biopsies and treatment monitoring. The present study investigated the dynamics of plasma HER2 gene copy number and amplification in...... cfDNA during neoadjuvant chemotherapy....

  3. The Clinical Utilization of Circulating Cell Free DNA (CCFDNA in Blood of Cancer Patients

    Directory of Open Access Journals (Sweden)

    Yanyuan Wu

    2013-09-01

    Full Text Available Qualitative and quantitative testing of circulating cell free DNA (CCFDNA can be applied for the management of malignant and benign neoplasms. Detecting circulating DNA in cancer patients may help develop a DNA profile for early stage diagnosis in malignancies. The technical issues of obtaining, using, and analyzing CCFDNA from blood will be discussed.

  4. Real-time PCR evaluation of cell-free DNA subjected to various storage and shipping conditions.

    Science.gov (United States)

    Wang, Q; Cai, Y; Brady, P; Vermeesch, J R

    2015-01-01

    In this study, we attempted to explore the factors affecting the yield of cell-free fetal DNA (cffDNA) obtained from maternal blood samples, including the use of different types of collection tubes, the interval between sample processing, and sample shipping under extreme weather conditions. Blood samples were drawn into K3EDTA tubes and cell-stabilizing tubes (Streck blood collection tube, BCT) from women pregnant with male fetuses. Real time PCR was used to amplify a β-actin gene fragment to measure the total plasma cell-free DNA concentration, while an SRY gene fragment was used to quantify the cffDNA. The samples in the K3EDTA tubes revealed a decreased quantity of SRY after 5 days of transportation, with a median of 25.9 copies/mL (P tubes. We observed a statistically significant increase in stability of the amount of total DNA in the blood samples stored in K3EDTA tubes (P tubes. PMID:26505430

  5. Non-random fragmentation patterns in circulating cell-free DNA reflect epigenetic regulation

    OpenAIRE

    Ivanov, Maxim; Baranova, Ancha; Butler, Timothy; Spellman, Paul; Mileyko, Vladislav

    2015-01-01

    Background The assessment of cell-free circulating DNA fragments, also known as a "liquid biopsy" of the patient's plasma, is an important source for the discovery and subsequent non-invasive monitoring of cancer and other pathological conditions. Although the nucleosome-guided fragmentation patterns of cell-free DNA (cfDNA) have not yet been studied in detail, non-random representation of cfDNA sequencies may reflect chromatin features in the tissue of origin at gene-regulation level. Result...

  6. Adjustments to the preanalytical phase of quantitative cell-free DNA analysis

    OpenAIRE

    Abel Jacobus Bronkhorst; Janine Aucamp; Piet J. Pretorius

    2015-01-01

    Evaluating the kinetics of cell-free DNA (cfDNA) in the blood of cancer patients could be a strong auxiliary component to the molecular characterization of cfDNA, but its potential clinical significance is obscured by the absence of an analytical consensus. To utilize quantitative cfDNA assessment with confidence, it is crucial that the preanalytical phase is standardized. In a previous publication, several preanalytical variables that may affect quantitative measurements of cfDNA were identi...

  7. Accuracy of non-invasive prenatal testing using cell-free DNA for detection of Down, Edwards and Patau syndromes: a systematic review and meta-analysis

    OpenAIRE

    Taylor-Phillips, Sian; Freeman, Karoline; Geppert, Julia; Agbebiyi, Adeola; Olalekan A Uthman; Madan, Jason; Clarke, Angus; Quenby, Siobhan; Clarke, Aileen

    2016-01-01

    Objective To measure test accuracy of non-invasive prenatal testing (NIPT) for Down, Edwards and Patau syndromes using cell-free fetal DNA and identify factors affecting accuracy. Design Systematic review and meta-analysis of published studies. Data sources PubMed, Ovid Medline, Ovid Embase and the Cochrane Library published from 1997 to 9 February 2015, followed by weekly autoalerts until 1 April 2015. Eligibility criteria for selecting studies English language journal articles describing ca...

  8. Plasma Cell-Free DNA in Paediatric Lymphomas

    Science.gov (United States)

    Mussolin, Lara; Burnelli, Roberta; Pillon, Marta; Carraro, Elisa; Farruggia, Piero; Todesco, Alessandra; Mascarin, Maurizio; Rosolen, Angelo

    2013-01-01

    Background: Extracellular circulating DNA (cfDNA) can be found in small amounts in plasma of healthy individuals. Increased levels of cfDNA have been reported in patients with cancer of breast, cervix, colon, liver and it was shown that cfDNA can originate from both tumour and non-tumour cells. Objectives: Levels of cfDNA of a large series of children with lymphoma were evaluated and analyzed in relation with clinical characteristics. Methods: plasma cfDNA levels obtained at diagnosis in 201 paediatric lymphoma patients [43 Hodgkin lymphomas (HL), 45 anaplastic large cell lymphomas (ALCL), 88 Burkitt lymphomas (BL), 17 lymphoblastic (LBL), 8 diffuse large B cell lymphoma (DLBCL)] and 15 healthy individuals were determined using a quantitative PCR assay for POLR2 gene and, in addition, for NPM-ALK fusion gene in ALCL patients. Wilcoxon rank sum test was used to compare plasma levels among different patient subgroups and controls and to analyze relationship between levels of cfDNA and clinical characteristics. Results: Levels of cfDNA in lymphoma patients were significantly higher compared with controls (p<0.0001). CfDNA was associated with median age (p=0.01) in HL, and with stage in ALCL (p=0.01). In HL patients high cfDNA levels were correlated with poor prognosis (p=0.03). In ALCL we found that most of the cfDNA (77%) was non-tumor DNA. Conclusion: level of plasma cfDNA might constitute an important non-invasive tool at diagnosis in lymphoma patients' management; in particular in patients with HL, cfDNA seems to be a promising prognostic biomarker. PMID:23678368

  9. Value of urinary topoisomerase-IIA cell-free DNA for diagnosis of bladder cancer

    Science.gov (United States)

    Kim, Ye-Hwan; Yan, Chunri; Lee, Il-Seok; Piao, Xuan-Mei; Byun, Young Joon; Jeong, Pildu; Kim, Won Tae; Yun, Seok-Joong

    2016-01-01

    Purpose Topoisomerase-II alpha (TopoIIA ), a DNA gyrase isoform that plays an important role in the cell cycle, is present in normal tissues and various human cancers, and can show altered expression in both. The aim of the current study was to examine the value of urinary TopoIIA cell-free DNA as a noninvasive diagnosis of bladder cancer (BC). Materials and Methods Two patient cohorts were examined. Cohort 1 (73 BC patients and seven controls) provided bladder tissue samples, whereas cohort 2 (83 BC patients, 54 nonmalignant hematuric patients, and 61 normal controls) provided urine samples. Real-time quantitative polymerase chain reaction was used to measure expression of TopoIIA mRNA in tissues and TopoIIA cell-free DNA in urine samples. Results The results showed that expression of TopoIIA mRNA in BC tissues was significantly higher than that in noncancer control tissues (p<0.001). The expression of urinary TopoIIA cell-free DNA in BC patients was also significantly higher than that in noncancer patient controls and hematuria patients (p < 0.001 and p < 0.001, respectively). High expression of urinary TopoIIA cell-free DNA was also detected in muscle invasive bladder cancer (MIBC) when compared with nonmuscle invasive bladder cancer (NMIBC) (p=0.002). Receiver operating characteristics (ROC) curve analysis was performed to examine the sensitivity/specificity of urinary TopoIIA cell-free DNA for diagnosing BC, NMIBC, and MIBC. The areas under the ROC curve for BC, NMIBC, and MIBC were 0.741, 0.701, and 0.838, respectively. Conclusions In summary, the results of this study provide evidence that cell-free TopoIIA DNA may be a potential biomarker for BC. PMID:26981592

  10. Elevated levels of cell-free circulating DNA in patients with acute dengue virus infection.

    Directory of Open Access Journals (Sweden)

    Tran Thi Ngoc Ha

    Full Text Available BACKGROUND: Apoptosis is thought to play a role in the pathogenesis of severe dengue and the release of cell-free DNA into the circulatory system in several medical conditions. Therefore, we investigated circulating DNA as a potential biomarker for severe dengue. METHODS AND FINDINGS: A direct fluorometric degradation assay using PicoGreen was performed to quantify cell-free DNA from patient plasma. Circulating DNA levels were significantly higher in patients with dengue virus infection than with other febrile illnesses and healthy controls. Remarkably, the increase of DNA levels correlated with the severity of dengue. Additionally, multivariate logistic regression analysis showed that circulating DNA levels independently correlated with dengue shock syndrome. CONCLUSIONS: Circulating DNA levels were increased in dengue patients and correlated with dengue severity. Additional studies are required to show the benefits of this biomarker in early dengue diagnosis and for the prognosis of shock complication.

  11. Adjustments to the preanalytical phase of quantitative cell-free DNA analysis

    Directory of Open Access Journals (Sweden)

    Abel Jacobus Bronkhorst

    2016-03-01

    Full Text Available Evaluating the kinetics of cell-free DNA (cfDNA in the blood of cancer patients could be a strong auxiliary component to the molecular characterization of cfDNA, but its potential clinical significance is obscured by the absence of an analytical consensus. To utilize quantitative cfDNA assessment with confidence, it is crucial that the preanalytical phase is standardized. In a previous publication, several preanalytical variables that may affect quantitative measurements of cfDNA were identified, and the most confounding variables were assessed further using the growth medium of cultured cancer cells as a source of cfDNA (“Cell-free DNA: Preanalytical variables” [1]. The data accompanying this report relates to these experiments, which includes numerous changes to the sample handling and isolation protocols, and can be used for the interpretation of these results and other similar experiments by different researchers.

  12. Adjustments to the preanalytical phase of quantitative cell-free DNA analysis

    Science.gov (United States)

    Bronkhorst, Abel Jacobus; Aucamp, Janine; Pretorius, Piet J.

    2015-01-01

    Evaluating the kinetics of cell-free DNA (cfDNA) in the blood of cancer patients could be a strong auxiliary component to the molecular characterization of cfDNA, but its potential clinical significance is obscured by the absence of an analytical consensus. To utilize quantitative cfDNA assessment with confidence, it is crucial that the preanalytical phase is standardized. In a previous publication, several preanalytical variables that may affect quantitative measurements of cfDNA were identified, and the most confounding variables were assessed further using the growth medium of cultured cancer cells as a source of cfDNA (“Cell-free DNA: Preanalytical variables” [1]). The data accompanying this report relates to these experiments, which includes numerous changes to the sample handling and isolation protocols, and can be used for the interpretation of these results and other similar experiments by different researchers. PMID:26862578

  13. Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma

    OpenAIRE

    Philip Burnham; Min Seong Kim; Sean Agbor-Enoh; Helen Luikart; Hannah A. Valantine; Kiran K Khush; Iwijn De Vlaminck

    2016-01-01

    Circulating cell-free DNA (cfDNA) is emerging as a powerful monitoring tool in cancer, pregnancy and organ transplantation. Nucleosomal DNA, the predominant form of plasma cfDNA, can be adapted for sequencing via ligation of double-stranded DNA (dsDNA) adapters. dsDNA library preparations, however, are insensitive to ultrashort, degraded cfDNA. Drawing inspiration from advances in paleogenomics, we have applied a single-stranded DNA (ssDNA) library preparation method to sequencing of cfDNA in...

  14. Cell-free DNA for diagnosing myocardial infarction: not ready for prime time.

    Science.gov (United States)

    Lippi, Giuseppe; Sanchis-Gomar, Fabian; Cervellin, Gianfranco

    2015-11-01

    A modest amount of cell-free DNA is constantly present in human blood, originating from programmed cell death, apoptosis and rupture of blood cells or pathogens. Acute or chronic cell injury contributes to enhance the pool of circulating nucleic acids, so that their assessment may be regarded as an appealing perspective for diagnosing myocardial ischemia. We performed a search in Medline, Web of Science and Scopus to identify clinical studies that investigated the concentration of cell-free DNA in patients with myocardial ischemia. Overall, eight case-control studies could be detected and reviewed. Although the concentration of cell-free DNA was found to be higher in the diseased than in the healthy population, the scenario was inconclusive due to the fact that the overall number of subjects studied was modest, the populations were unclearly defined, cases and controls were not adequately matched, the methodology for measuring the reference cardiac biomarkers was inadequately described, and the diagnostic performance of cell-free DNA was not benchmarked against highly sensitive troponin immunoassays. Several biological and technical hurdles were also identified in cell-free DNA testing, including the lack of specificity and unsuitable kinetics for early diagnosis of myocardial ischemia, the long turnaround time and low throughput, the need for specialized instrumentation and dedicated personnel, the lack of standardization or harmonization of analytical techniques, the incremental costs and the high vulnerability to preanalytical variables. Hence it seems reasonable to conclude that the analysis of cell-free DNA is not ready for prime time in diagnostics of myocardial ischemia. PMID:25883207

  15. 孕妇外周血中游离胎儿DNA检测在诊断胎儿染色体异常中的应用价值%Value of detection of cell-free fetal DNA in maternal plasma in the prenatal diagnosis of chromosomal abnormalities

    Institute of Scientific and Technical Information of China (English)

    汪淑娟; 高志英; 卢彦平; 李亚里; 游艳琴; 张立文; 汪龙霞; 徐虹

    2012-01-01

    细胞占羊水总细胞的2%,未行引产,胎儿出生后未发现结构异常;有l例因在穿刺手术前已发生胎死宫内,未能进行核型验证,其前超声提示胎儿较孕周小3周,且全身水肿.(3)3组孕妇血浆中游离胎儿DNA检测结果阴性者3173例,经电话随访,截止至2012年5月30日,已有1230例新生儿出生,经检查均未发现唐氏综合征患儿.结论 游离胎儿DNA检测是一种安全、准确、高通量的21三体产前检测方法,与染色体核型分析结果相符;作为唐氏综合征患儿血清学筛查高风险孕妇的进一步筛选方法,可大幅度减少介入性产前诊断,并可作为临床诊断唐氏综合征的依据.%Objective To investigate the value of detection of fetal cell-free fetal DNA(cff-DNA)in maternal plasma in the prenatal diagnosis of chromosomal abnormalities.Methods The plasma from 3200 gravidas(singleton with 20.3 ± 3.8 gestational weeks)was collected from April 1st 2011 to May 30th 2012.They were divided into 3 groups:(1)To tally 1720 cases were included in the high-risk serological screening group,in which women were younger than 35 years and got high-risk results in serological screening;(2)To tally 1310 cases were included in the advanced age group,in which women's age was more than 35 years;(3)To tally 170 cases were included in the supplementary group,in which women were younger than 35 years and got low-risk results in serological screening,or women who didn't take serological screening tests.All the 3030 gravidas in group 1 and 2 didn't take invasive prenatal diagnosis because of fear of abortion or short of prenatal diagnosis.Cff-DNA were detected by next generation sequencing in Shenzhen BGI Genomics Center for clinical laboratory.Amniocentesis and karyotype analysis were provided to the positive cases and women with negative results were followed-up by telephone.Results(1)The 3200 cases took cff-DNA detection,and 31 cases got positive results,including 27 cases of trisomy 21 and 4 cases of

  16. Processing of DNA for nonhomologous end-joining by cell-free extract

    OpenAIRE

    Budman, Joe; Chu, Gilbert

    2005-01-01

    In mammalian cells, nonhomologous end-joining (NHEJ) repairs DNA double-strand breaks created by ionizing radiation and V(D)J recombination. We have developed a cell-free system capable of processing and joining noncompatible DNA ends. The system had key features of NHEJ in vivo, including dependence on Ku, DNA-PKcs, and XRCC4/Ligase4. The NHEJ reaction had striking properties. Processing of noncompatible ends involved polymerase and nuclease activities that often stabilized the alignment of ...

  17. Synthesis and cell-free cloning of DNA libraries using programmable microfluidics

    OpenAIRE

    Yehezkel, Tuval Ben; Rival, Arnaud; Raz, Ofir; Cohen, Rafael; Marx, Zipora; Camara, Miguel; Dubern, Jean-Frédéric; Koch, Birgit; Heeb, Stephan; Krasnogor, Natalio; Delattre, Cyril; Shapiro, Ehud

    2015-01-01

    Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and a...

  18. Circulating cell free DNA as a predictor of systemic lupus erythematosus severity and monitoring of therapy

    OpenAIRE

    Olfat M. Hendy; Tawfik Abdel Motalib; Mona A. El Shafie; Fatma A. Khalaf; Sobhy E. Kotb; Aziza Khalil; Salwa R. Ali

    2016-01-01

    Background: Systemic lupus erythematosus (SLE) is the most heterogeneous chronic autoimmune disease; it is characterized by the presence of auto reactive B and T cells, responsible for the aberrant production of a broad and heterogeneous group of autoantibodies. Recent studies using various detection methods have demonstrated the elevations of circulating DNA in SLE patients. Aim of the study: The current study aimed to measure cell-free DNA (cf-DNA) in SLE patients as a potential tool to ...

  19. Tumor-Related Methylated Cell-Free DNA and Circulating Tumor Cells in Melanoma

    OpenAIRE

    Salvianti, Francesca; Orlando, Claudio; Massi, Daniela; DE GIORGI, VINCENZO; Grazzini, Marta; Pazzagli, Mario; Pinzani, Pamela

    2016-01-01

    Solid tumor release into the circulation cell-free DNA (cfDNA) and circulating tumor cells (CTCs) which represent promising biomarkers for cancer diagnosis. Circulating tumor DNA may be studied in plasma from cancer patients by detecting tumor specific alterations, such as genetic or epigenetic modifications. Ras association domain family 1 isoform A (RASSF1A) is a tumor suppressor gene silenced by promoter hypermethylation in a variety of human cancers including melanoma. The aim of the pres...

  20. Construction of a Sequencing Library from Circulating Cell-Free DNA.

    Science.gov (United States)

    Fang, Nan; Löffert, Dirk; Akinci-Tolun, Rumeysa; Heitz, Katja; Wolf, Alexander

    2016-01-01

    Circulating DNA is cell-free DNA (cfDNA) in serum or plasma that can be used for non-invasive prenatal testing, as well as cancer diagnosis, prognosis, and stratification. High-throughput sequence analysis of the cfDNA with next-generation sequencing technologies has proven to be a highly sensitive and specific method in detecting and characterizing mutations in cancer and other diseases, as well as aneuploidy during pregnancy. This unit describes detailed procedures to extract circulating cfDNA from human serum and plasma and generate sequencing libraries from a wide concentration range of circulating DNA. © 2016 by John Wiley & Sons, Inc. PMID:27038390

  1. Replication independent formation of extrachromosomal circular DNA in mammalian cell-free system.

    Directory of Open Access Journals (Sweden)

    Zoya Cohen

    Full Text Available Extrachromosomal circular DNA (eccDNA is a pool of circular double stranded DNA molecules found in all eukaryotic cells and composed of repeated chromosomal sequences. It was proposed to be involved in genomic instability, aging and alternative telomere lengthening. Our study presents novel mammalian cell-free system for eccDNA generation. Using purified protein extract we show that eccDNA formation does not involve de-novo DNA synthesis suggesting that eccDNA is generated through excision of chromosomal sequences. This process is carried out by sequence-independent enzymes as human protein extract can produce mouse-specific eccDNA from high molecular weight mouse DNA, and vice versa. EccDNA production does not depend on ATP, requires residual amounts of Mg(2+ and is enhanced by double strand DNA breaks.

  2. High recovery of cell-free methylated DNA based on a rapid bisulfite-treatment protocol

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    Pedersen Inge

    2012-03-01

    Full Text Available Abstract Background Detection of cell-free methylated DNA in plasma is a promising tool for tumour diagnosis and monitoring. Due to the very low amounts of cell-free DNA in plasma, analytical sensitivity is of utmost importance. The vast majority of currently available methods for analysing DNA methylation are based on bisulfite-mediated deamination of cytosine. Cytosine is rapidly converted to uracil during bisulfite treatment, whereas 5-methylcytosine is only slowly converted. Hence, bisulfite treatment converts an epigenetic modification into a difference in sequence, amenable to analysis either by sequencing or PCR based methods. However, the recovery of bisulfite-converted DNA is very poor. Results Here we introduce an alternative method for the crucial steps of bisulfite treatment with high recovery. The method is based on an accelerated deamination step and alkaline desulfonation in combination with magnetic silica purification of DNA, allowing preparation of deaminated DNA from patient samples in less than 2 hours. Conclusions The method presented here allows low levels of DNA to be easily and reliably analysed, a prerequisite for the clinical usefulness of cell-free methylated DNA detection in plasma.

  3. An Economic Analysis of Cell-Free DNA Non-Invasive Prenatal Testing in the US General Pregnancy Population.

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    Peter Benn

    Full Text Available Analyze the economic value of replacing conventional fetal aneuploidy screening approaches with non-invasive prenatal testing (NIPT in the general pregnancy population.Using decision-analysis modeling, we compared conventional screening to NIPT with cell-free DNA (cfDNA analysis in the annual US pregnancy population. Sensitivity and specificity for fetal aneuploidies, trisomy 21, trisomy 18, trisomy 13, and monosomy X, were estimated using published data and modeling of both first- and second trimester screening. Costs were assigned for each prenatal test component and for an affected birth. The overall cost to the healthcare system considered screening costs, the number of aneuploid cases detected, invasive procedures performed, procedure-related euploid losses, and affected pregnancies averted. Sensitivity analyses evaluated the effect of variation in parameters. Costs were reported in 2014 US Dollars.Replacing conventional screening with NIPT would reduce healthcare costs if it can be provided for $744 or less in the general pregnancy population. The most influential variables were timing of screening entry, screening costs, and pregnancy termination rates. Of the 13,176 affected pregnancies undergoing screening, NIPT detected 96.5% (12,717/13,176 of cases, compared with 85.9% (11,314/13,176 by conventional approaches. NIPT reduced invasive procedures by 60.0%, with NIPT and conventional methods resulting in 24,596 and 61,430 invasive procedures, respectively. The number of procedure-related euploid fetal losses was reduced by 73.5% (194/264 in the general screening population.Based on our analysis, universal application of NIPT would increase fetal aneuploidy detection rates and can be economically justified. Offering this testing to all pregnant women is associated with substantial prenatal healthcare benefits.

  4. An Economic Analysis of Cell-Free DNA Non-Invasive Prenatal Testing in the US General Pregnancy Population

    Science.gov (United States)

    Benn, Peter; Curnow, Kirsten J.; Chapman, Steven; Michalopoulos, Steven N.; Hornberger, John; Rabinowitz, Matthew

    2015-01-01

    Objective Analyze the economic value of replacing conventional fetal aneuploidy screening approaches with non-invasive prenatal testing (NIPT) in the general pregnancy population. Methods Using decision-analysis modeling, we compared conventional screening to NIPT with cell-free DNA (cfDNA) analysis in the annual US pregnancy population. Sensitivity and specificity for fetal aneuploidies, trisomy 21, trisomy 18, trisomy 13, and monosomy X, were estimated using published data and modeling of both first- and second trimester screening. Costs were assigned for each prenatal test component and for an affected birth. The overall cost to the healthcare system considered screening costs, the number of aneuploid cases detected, invasive procedures performed, procedure-related euploid losses, and affected pregnancies averted. Sensitivity analyses evaluated the effect of variation in parameters. Costs were reported in 2014 US Dollars. Results Replacing conventional screening with NIPT would reduce healthcare costs if it can be provided for $744 or less in the general pregnancy population. The most influential variables were timing of screening entry, screening costs, and pregnancy termination rates. Of the 13,176 affected pregnancies undergoing screening, NIPT detected 96.5% (12,717/13,176) of cases, compared with 85.9% (11,314/13,176) by conventional approaches. NIPT reduced invasive procedures by 60.0%, with NIPT and conventional methods resulting in 24,596 and 61,430 invasive procedures, respectively. The number of procedure-related euploid fetal losses was reduced by 73.5% (194/264) in the general screening population. Conclusion Based on our analysis, universal application of NIPT would increase fetal aneuploidy detection rates and can be economically justified. Offering this testing to all pregnant women is associated with substantial prenatal healthcare benefits. PMID:26158465

  5. Methylation of cell-free circulating DNA in the diagnosis of cancer

    OpenAIRE

    Warton, Kristina; Samimi, Goli

    2015-01-01

    A range of molecular alterations found in tumor cells, such as DNA mutations and DNA methylation, is reflected in cell-free circulating DNA (circDNA) released from the tumor into the blood, thereby making circDNA an ideal candidate for the basis of a blood-based cancer diagnosis test. In many cancer types, mutations driving tumor development and progression are present in a wide range of oncogenes and tumor suppressor genes. However, even when a gene is consistently mutated in a particular ca...

  6. Kinetics of Circulating Plasma Cell-Free DNA in Paediatric Classical Hodgkin Lymphoma

    OpenAIRE

    Primerano, Simona; Burnelli, Roberta; Carraro, Elisa; Pillon, Marta; Elia, Caterina; Farruggia, Piero; Sala, Alessandra; Vinti, Luciana; Buffardi, Salvatore; Basso, Giuseppe; Mascarin, Maurizio; Mussolin, Lara

    2016-01-01

    Levels of plasma cell-free DNA (cfDNA) of a large series of children with classical Hodgkin lymphoma (cHL) were evaluated and analyzed at diagnosis and during chemotherapy treatment in relation with clinical characteristics. CfDNA levels in cHL patients were significantly higher compared with controls (p=0.002). CfDNA at diagnosis was correlated with presence of B symptoms (p=0.027) and high erythrocyte sedimentation rate (p=0.049). We found that the increasing of plasma cfDNA after first che...

  7. Synthesis and cell-free cloning of DNA libraries using programmable microfluidics.

    Science.gov (United States)

    Ben Yehezkel, Tuval; Rival, Arnaud; Raz, Ofir; Cohen, Rafael; Marx, Zipora; Camara, Miguel; Dubern, Jean-Frédéric; Koch, Birgit; Heeb, Stephan; Krasnogor, Natalio; Delattre, Cyril; Shapiro, Ehud

    2016-02-29

    Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development. PMID:26481354

  8. Synthesis and cell-free cloning of DNA libraries using programmable microfluidics

    Science.gov (United States)

    Yehezkel, Tuval Ben; Rival, Arnaud; Raz, Ofir; Cohen, Rafael; Marx, Zipora; Camara, Miguel; Dubern, Jean-Frédéric; Koch, Birgit; Heeb, Stephan; Krasnogor, Natalio; Delattre, Cyril; Shapiro, Ehud

    2016-01-01

    Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development. PMID:26481354

  9. Non-Invasive Prenatal Testing Using Cell Free DNA in Maternal Plasma: Recent Developments and Future Prospects

    Directory of Open Access Journals (Sweden)

    Peter Benn

    2014-05-01

    Full Text Available Recent advances in molecular genetic technologies have facilitated non-invasive prenatal testing (NIPT through the analysis of cell-free fetal DNA in maternal plasma. NIPT can be used to identify monogenic disorders including the identification of autosomal recessive disorders where the maternally inherited mutation needs to be identified in the presence of an excess of maternal DNA that contains the same mutation. In the future, simultaneous screening for multiple monogenic disorders is anticipated. Several NIPT methods have been developed to screen for trisomy. These have been shown to be effective for fetal trisomy 21, 18 and 13. Although the testing has been extended to sex chromosome aneuploidy, robust estimates of the efficacy are not yet available and maternal mosaicism for gain or loss of an X-chromosome needs to be considered. Using methods based on the analysis of single nucleotide polymorphisms, diandric triploidy can be identified. NIPT is being developed to identify a number of microdeletion syndromes including α-globin gene deletion. NIPT is a profoundly important development in prenatal care that is substantially advancing the individual patient and public health benefits achieved through conventional prenatal screening and diagnosis.

  10. Circulating Cell-Free DNA Enables Noninvasive Diagnosis of Heart Transplant Rejection

    OpenAIRE

    De Vlaminck, Iwijn; Valantine, Hannah A.; Snyder, Thomas M.; Strehl, Calvin; Cohen, Garrett; Luikart, Helen; Neff, Norma F.; Okamoto, Jennifer; Bernstein, Daniel; Weisshaar, Dana; Quake, Stephen R.; Khush, Kiran K.

    2014-01-01

    Monitoring allograft health is an important component of posttransplant therapy. Endomyocardial biopsy is the current gold standard for cardiac allograft monitoring but is an expensive and invasive procedure. Proof of principle of a universal, noninvasive diagnostic method based on high-throughput screening of circulating cell-free donor-derived DNA (cfdDNA) was recently demonstrated in a small retrospective cohort. We present the results of a prospective cohort study (65 patients, 565 sample...

  11. Circulating cell-free DNA and its integrity as a prognostic marker for breast cancer

    OpenAIRE

    Iqbal, Sobuhi; Vishnubhatla, Sreenivas; Raina, Vinod; Sharma, Surabhi; Gogia, Ajay; Suryanarayana S.V. Deo; Mathur, Sandeep; Shukla, Nutan Kumar

    2015-01-01

    The aim of our study was to look for alternative predictive biomarkers for breast cancer management in limited resource setup. A comprehensive analysis of circulating cell-free DNA (CCFD) in serum at baseline was performed to assess its prognostic potential. Quantitative polymerase chain reaction (qPCR) of ALU sequences using ALU115 and ALU247 primers was carried out in patients (N: baseline 148, postoperative 47) and 51 healthy controls. Mean serum DNA integrity, levels of ALU 247 and levels...

  12. Monitoring of organ transplants through genomic analyses of circulating cell-free DNA

    Science.gov (United States)

    de Vlaminck, Iwijn

    Solid-organ transplantation is the preferred treatment for patients with end-stage organ diseases, but complications due to infection and acute rejection undermine its long-term benefits. While clinicians strive to carefully monitor transplant patients, diagnostic options are currently limited. My colleagues and I in the lab of Stephen Quake have found that a combination of next-generation sequencing with a phenomenon called circulating cell-free DNA enables non-invasive diagnosis of both infection and rejection in transplantation. A substantial amount of small fragments of cell-free DNA circulate in blood that are the debris of dead cells. We discovered that donor specific DNA is released in circulation during injury to the transplant organ and we show that the proportion of donor DNA in plasma is predictive of acute rejection in heart and lung transplantation. We profiled viral and bacterial DNA sequences in plasma of transplant patients and discovered that the relative representation of different viruses and bacteria is informative of immunosuppression. This discovery suggested a novel biological measure of a person's immune strength, a finding that we have more recently confirmed via B-cell repertoire sequencing. Lastly, our studies highlight applications of shotgun sequencing of cell-free DNA in the broad, hypothesis free diagnosis of infection.

  13. Admission Cell Free DNA as a Prognostic Factor in Burns: Quantification by Use of a Direct Rapid Fluorometric Technique

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    Yaron Shoham

    2014-01-01

    Full Text Available Background. Despite great advances in the treatment of burn patients, useful prognostic markers are sparse. During the past years there has been increasing interest in circulating plasma cell free DNA as a potential marker for tissue injury. We have developed a rapid direct fluorescent assay for cell free DNA quantification that allows obtaining accurate, fast, and inexpensive measurements. Objective. To use this technique for measuring plasma cell free DNA levels in burn patients and to further explore the use of cell free DNA as a potential marker of patient outcome in burns. Methods. Cell free DNA levels obtained from 14 burn victims within 6 hours of injury and 14 healthy controls were quantified by a direct rapid fluorometric assay. Results. Patient admission cell free DNA levels were significantly elevated compared with that of controls (1797 ± 1523 ng/mL versus 374 ± 245 ng/mL, P=0.004. There are statistically significant correlations between cell free DNA admission levels and burn degree (Spearman’s correlation = 0.78, P=0.001, total body surface area (Spearman’s correlation = 0.61, P=0.02, and total burn volume (Spearman’s correlation = 0.64, P=0.014. Conclusions. Admission cell free DNA levels can serve as a prognostic factor in burns and future routine use can be made possible by use of our direct rapid fluorometric assay.

  14. A modified Phenol-chloroform extraction method for isolating circulating cell free DNA of tumor patients

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    Clemens Hufnagl

    2013-03-01

    Full Text Available Searching for new cancer biomarkers, circulating cell-free DNA (cfDNA has become an appealing target of interest as an elevated level of cfDNA has been detected in the circulation of cancer patients in comparison with healthy controls. Since cfDNA can be isolated from the circulation and other body fluids of patients without harming their physical condition, cfDNA is becoming a promising candidate as a novel non-invasive biomarker for cancer. The challenge in the diagnostic analysis of cfDNA is its very low presence in human plasma/serum and its partially strong fragmentation. Here we evaluated a modified phenol/chloroform extraction method for the isolation of cfDNA and compared it with published standard methods for cfDNA isolation.

  15. Droplet Digital PCR combined with minisequencing, a new approach to analyze fetal DNA from maternal blood: application to the non-invasive prenatal diagnosis of achondroplasia.

    OpenAIRE

    Orhant, Lucie; Anselem, Olivia; Fradin, Mélanie; Becker, Pierre Hadrien; Beugnet, Caroline; Deburgrave, Nathalie; Tafuri, Gilles; Letourneur, Franck; Goffinet, François; Khattabi, Laïla Allach El; Leturcq, France; Bienvenu, Thierry; Tsatsaris, Vassilis; Nectoux, Juliette

    2016-01-01

    Achondroplasia is generally detected by abnormal prenatal ultrasound-findings in the third trimester of pregnancy, and then confirmed by molecular genetic testing of fetal genomic DNA obtained by aspiration of amniotic fluid. This invasive procedure presents a small but significant risk for both the fetus and mother. Therefore, non-invasive procedures using cell-free-fetal DNA in maternal plasma have been developed for the detection of the fetal achondroplasia mutations.MethodsTo determine wh...

  16. Diagnosi prenatale non invasiva di malattie monogeniche attraverso la ricerca e l'isolamento di cellule e DNA fetale nel sangue materno

    OpenAIRE

    Contini, Antonella

    2012-01-01

    Prenatal genetic diagnosis of monogenic diseases and chromosomal abnormalities is usually performed collecting fetal samples through villocentesis or amniocentesis. These invasive procedures are associated with 0.5-1% risk for the fetus. Due to it, in recent years, much effort has been made to develop non invasive prenatal diagnosis (NIPD). Two potential non invasive approaches involve the analysis of fetal cells and cell-free fetal DNA (cffDNA) found in the maternal circulation. The prese...

  17. Cell-free (RNA and cell-associated (DNA HIV-1 and postnatal transmission through breastfeeding.

    Directory of Open Access Journals (Sweden)

    James Ndirangu

    Full Text Available INTRODUCTION: Transmission through breastfeeding remains important for mother-to-child transmission (MTCT in resource-limited settings. We quantify the relationship between cell-free (RNA and cell-associated (DNA shedding of HIV-1 virus in breastmilk and the risk of postnatal HIV-1 transmission in the first 6 months postpartum. MATERIALS AND METHODS: Thirty-six HIV-positive mothers who transmitted HIV-1 by breastfeeding were matched to 36 non-transmitting HIV-1 infected mothers in a case-control study nested in a cohort of HIV-infected women. RNA and DNA were quantified in the same breastmilk sample taken at 6 weeks and 6 months. Cox regression analysis assessed the association between cell-free and cell-associated virus levels and risk of postnatal HIV-1 transmission. RESULTS: There were higher median levels of cell-free than cell-associated HIV-1 virus (per ml in breastmilk at 6 weeks and 6 months. Multivariably, adjusting for antenatal CD4 count and maternal plasma viral load, at 6 weeks, each 10-fold increase in cell-free or cell-associated levels (per ml was significantly associated with HIV-1 transmission but stronger for cell-associated than cell-free levels [2.47 (95% CI 1.33-4.59 vs. aHR 1.52 (95% CI, 1.17-1.96, respectively]. At 6 months, cell-free and cell-associated levels (per ml in breastmilk remained significantly associated with HIV-1 transmission but was stronger for cell-free than cell-associated levels [aHR 2.53 (95% CI 1.64-3.92 vs. 1.73 (95% CI 0.94-3.19, respectively]. CONCLUSIONS: The findings suggest that cell-associated virus level (per ml is more important for early postpartum HIV-1 transmission (at 6 weeks than cell-free virus. As cell-associated virus levels have been consistently detected in breastmilk despite antiretroviral therapy, this highlights a potential challenge for resource-limited settings to achieve the UNAIDS goal for 2015 of eliminating vertical transmission. More studies would further knowledge on

  18. Cell-free DNA in the Cerebrospinal Fluid under Emotional Stress

    OpenAIRE

    Mariia Zharova; Pavel Umriukhin; Natalia Veiko

    2016-01-01

    Background: Cell free circulating DNA (cfDNA) in blood is known to be a tumor marker however there is no information about its concentration in cerebrospinal fluid (CSF) in control and in emotional stress (ES). The aim of the study was to determine level of cfDNA in CSF of rats with different resistance to stress before and after ES. Methods: A total of 19 male Wistar rats weighing 200-220 g were included in this study. All rats were divided into 2 groups depending on the motor activity: acti...

  19. Cell-free DNA next-generation sequencing in pancreatobiliary carcinomas

    OpenAIRE

    Zill, Oliver A.; Greene, Claire; Sebisanovic, Dragan; Siew, LaiMun; Leng, Jim; Vu, Mary; HENDIFAR, ANDREW E.; Zhen WANG; Atreya, Chloe E.; Kelley, Robin K.; Van Loon, Katherine; Ko, Andrew H.; Tempero, Margaret A.; Bivona, Trever G; Munster, Pamela N.

    2015-01-01

    Patients with pancreatic and biliary carcinomas lack personalized treatment options, in part because biopsies are often inadequate for molecular characterization. Cell-free DNA (cfDNA) sequencing may enable a precision oncology approach in this setting. We attempted to prospectively analyze 54 genes in tumor and cfDNA for 26 patients. Tumor sequencing failed in nine patients (35%). In the remaining 17, 90.3% (95% CI: 73.1–97.5%) of mutations detected in tumor biopsies were also detected in cf...

  20. Cell-Free DNA as a Diagnostic Tool for Human Parasitic Infections.

    Science.gov (United States)

    Weerakoon, Kosala G; McManus, Donald P

    2016-05-01

    Parasites often cause devastating diseases and represent a significant public health and economic burden. More accurate and convenient diagnostic tools are needed in support of parasite control programmes in endemic regions, and for rapid point-of-care diagnosis in nonendemic areas. The detection of cell-free DNA (cfDNA) is a relatively new concept that is being applied in the current armamentarium of diagnostics. Here, we review the application of cfDNA detection with nucleic acid amplification tests for the diagnosis and evaluation of different human parasitic infections and highlight the significant benefits of the approach using non-invasive clinical samples. PMID:26847654

  1. DNA Methylation Landscapes of Human Fetal Development.

    Science.gov (United States)

    Slieker, Roderick C; Roost, Matthias S; van Iperen, Liesbeth; Suchiman, H Eka D; Tobi, Elmar W; Carlotti, Françoise; de Koning, Eelco J P; Slagboom, P Eline; Heijmans, Bastiaan T; Chuva de Sousa Lopes, Susana M

    2015-10-01

    Remodelling the methylome is a hallmark of mammalian development and cell differentiation. However, current knowledge of DNA methylation dynamics in human tissue specification and organ development largely stems from the extrapolation of studies in vitro and animal models. Here, we report on the DNA methylation landscape using the 450k array of four human tissues (amnion, muscle, adrenal and pancreas) during the first and second trimester of gestation (9,18 and 22 weeks). We show that a tissue-specific signature, constituted by tissue-specific hypomethylated CpG sites, was already present at 9 weeks of gestation (W9). Furthermore, we report large-scale remodelling of DNA methylation from W9 to W22. Gain of DNA methylation preferentially occurred near genes involved in general developmental processes, whereas loss of DNA methylation mapped to genes with tissue-specific functions. Dynamic DNA methylation was associated with enhancers, but not promoters. Comparison of our data with external fetal adrenal, brain and liver revealed striking similarities in the trajectory of DNA methylation during fetal development. The analysis of gene expression data indicated that dynamic DNA methylation was associated with the progressive repression of developmental programs and the activation of genes involved in tissue-specific processes. The DNA methylation landscape of human fetal development provides insight into regulatory elements that guide tissue specification and lead to organ functionality. PMID:26492326

  2. Circulating Cell-Free Tumour DNA in the Management of Cancer

    OpenAIRE

    Glenn Francis; Sandra Stein

    2015-01-01

    With the development of new sensitive molecular techniques, circulating cell-free tumour DNA containing mutations can be identified in the plasma of cancer patients. The applications of this technology may result in significant changes to the care and management of cancer patients. Whilst, currently, these “liquid biopsies” are used to supplement the histological diagnosis of cancer and metastatic disease, in the future these assays may replace the need for invasive procedures. Applications ...

  3. Kinetics of Circulating Plasma Cell-Free DNA in Paediatric Classical Hodgkin Lymphoma

    Science.gov (United States)

    Primerano, Simona; Burnelli, Roberta; Carraro, Elisa; Pillon, Marta; Elia, Caterina; Farruggia, Piero; Sala, Alessandra; Vinti, Luciana; Buffardi, Salvatore; Basso, Giuseppe; Mascarin, Maurizio; Mussolin, Lara

    2016-01-01

    Levels of plasma cell-free DNA (cfDNA) of a large series of children with classical Hodgkin lymphoma (cHL) were evaluated and analyzed at diagnosis and during chemotherapy treatment in relation with clinical characteristics. CfDNA levels in cHL patients were significantly higher compared with controls (p=0.002). CfDNA at diagnosis was correlated with presence of B symptoms (p=0.027) and high erythrocyte sedimentation rate (p=0.049). We found that the increasing of plasma cfDNA after first chemotherapy cycle seems to be associated with a worse prognosis (p=0.049). Levels of plasma cfDNA might constitute an interesting non-invasive tool in cHL patients' management. PMID:26918050

  4. Kinetics of Circulating Plasma Cell-Free DNA in Paediatric Classical Hodgkin Lymphoma.

    Science.gov (United States)

    Primerano, Simona; Burnelli, Roberta; Carraro, Elisa; Pillon, Marta; Elia, Caterina; Farruggia, Piero; Sala, Alessandra; Vinti, Luciana; Buffardi, Salvatore; Basso, Giuseppe; Mascarin, Maurizio; Mussolin, Lara

    2016-01-01

    Levels of plasma cell-free DNA (cfDNA) of a large series of children with classical Hodgkin lymphoma (cHL) were evaluated and analyzed at diagnosis and during chemotherapy treatment in relation with clinical characteristics. CfDNA levels in cHL patients were significantly higher compared with controls (p=0.002). CfDNA at diagnosis was correlated with presence of B symptoms (p=0.027) and high erythrocyte sedimentation rate (p=0.049). We found that the increasing of plasma cfDNA after first chemotherapy cycle seems to be associated with a worse prognosis (p=0.049). Levels of plasma cfDNA might constitute an interesting non-invasive tool in cHL patients' management. PMID:26918050

  5. Non-random fragmentation patterns in circulating cell-free DNA reflect epigenetic regulation

    Science.gov (United States)

    2015-01-01

    Background The assessment of cell-free circulating DNA fragments, also known as a "liquid biopsy" of the patient's plasma, is an important source for the discovery and subsequent non-invasive monitoring of cancer and other pathological conditions. Although the nucleosome-guided fragmentation patterns of cell-free DNA (cfDNA) have not yet been studied in detail, non-random representation of cfDNA sequencies may reflect chromatin features in the tissue of origin at gene-regulation level. Results In this study, we investigated the association between epigenetic landscapes of human tissues evident in the patterns of cfDNA in plasma by deep sequencing of human cfDNA samples. We have demonstrated that baseline characteristics of cfDNA fragmentation pattern are in concordance with the ones corresponding to cell lines-derived. To identify the loci differentially represented in cfDNA fragment, we mapped the transcription start sites within the sequenced cfDNA fragments and tested for association of these genomic coordinates with the relative strength and the patterns of gene expressions. Preselected sets of house-keeping and tissue specific genes were used as models for actively expressed and silenced genes. Developed measure of gene regulation was able to differentiate these two sets based on sequencing coverage near gene transcription start site. Conclusion Experimental outcomes suggest that cfDNA retains characteristics previously noted in genome-wide analysis of chromatin structure, in particular, in MNase-seq assays. Thus far the analysis of the DNA fragmentation pattern may aid further developing of cfDNA based biomarkers for a variety of human conditions. PMID:26693644

  6. Circulating Cell-Free DNA Enables Noninvasive Diagnosis of Heart Transplant Rejection

    Science.gov (United States)

    De Vlaminck, Iwijn; Valantine, Hannah A.; Snyder, Thomas M.; Strehl, Calvin; Cohen, Garrett; Luikart, Helen; Neff, Norma F.; Okamoto, Jennifer; Bernstein, Daniel; Weisshaar, Dana; Quake, Stephen R.; Khush, Kiran K.

    2014-01-01

    Monitoring allograft health is an important component of posttransplant therapy. Endomyocardial biopsy is the current gold standard for cardiac allograft monitoring but is an expensive and invasive procedure. Proof of principle of a universal, noninvasive diagnostic method based on high-throughput screening of circulating cell-free donor-derived DNA (cfdDNA) was recently demonstrated in a small retrospective cohort. We present the results of a prospective cohort study (65 patients, 565 samples) that tested the utility of cfdDNA in measuring acute rejection after heart transplantation. Circulating cell-free DNA was purified from plasma and sequenced (mean depth, 1.2 giga–base pairs) to quantify the fraction of cfdDNA. Through a comparison with endomyocardial biopsy results, we demonstrate that cfdDNA enables diagnosis of acute rejection after heart transplantation, with an area under the receiver operating characteristic curve of 0.83 and sensitivity and specificity that are comparable to the intrinsic performance of the biopsy itself. This noninvasive genome transplant dynamics approach is a powerful and informative method for routine monitoring of allograft health without incurring the risk, discomfort, and expense of an invasive biopsy. PMID:24944192

  7. Acceptance of non-invasive prenatal testing by cell free foetal DNA for foetal aneuploidy in a developing country: experience at a tertiary care centre in India

    OpenAIRE

    Namrata Kashyap; Mandakini Pradhan; Piyush Kumar; Neeta Singh

    2016-01-01

    Background: Non-invasive prenatal testing is a new technique which is deepening its root all over the world. Its tremendous potential lies in its ability of using cell free fetal DNA from the plasma of pregnant women. However, to what extent the technology has reached to a common person is also to be given a thought. hence the study was planned to assess the acceptability of non-invasive prenatal testing in Indian settings, to study about the awareness and baseline knowledge about Down's synd...

  8. Levels of cell-free DNA and plasma KRAS during treatment of advanced NSCLC

    DEFF Research Database (Denmark)

    Dowler Nygaard, Anneli; Spindler, Karen-Lise Garm; Pallisgaard, Niels;

    2014-01-01

    Non-small cell lung cancer (NSCLC) is one of the most common malignant tumours in the western world and is associated with a poor prognosis. Biomarkers predicting prognosis and therapeutic effects are highly required, and cell-free DNA (cfDNA) may be a feasible option. Genetic mutations can be...... analysed in plasma and may increase the scientific use of such measurements. In the present study, we investigated: i) the dynamics of cfDNA and plasma mutated KRAS (pmKRAS) during the treatment of patients with advanced NSCLC; and ii) the prognostic value of baseline cfDNA and pmKRAS. Sixty‑nine patients...... were included in a prospective biomarker trial. Inclusion criteria included advanced NSCLC, candidate for first-line treatment, no previous cancer within the five years prior to this study. Blood samples were drawn at baseline, day 8 and at progression. Analyses of cfDNA and KRAS mutations in plasma...

  9. PIK3CA mutation detection in metastatic biliary cancer using cell-free DNA

    OpenAIRE

    Kim, Seung Tae; Lira, Maruja; Deng, Shibing; Lee, Sujin; Park, Young Suk; Lim, Ho Yeong; Kang, Won Ki; Mao, Mao; Heo, Jin Seok; Kwon, Wooil; Jang, Kee-Taek; Lee, Jeeyun; Park, Joon Oh

    2015-01-01

    PIK3CA mutation is considered a good candidate for targeted therapies in cancers, especially biliary tract cancer (BTC). We evaluated the utility of cell free DNA (cfDNA) from serum by using droplet digital PCR (ddPCR) as an alternative source for PIK3CA mutation analysis. To identify matching archival tumour specimens from serum samples of advanced BTC patients, mutation detection using ddPCR with Bio-Rad's PrimePCR mutation and wild type assays were performed for PIK3CA p.E542K, p.E545K, an...

  10. Value of urinary topoisomerase-IIA cell-free DNA for diagnosis of bladder cancer

    OpenAIRE

    Kim, Ye-Hwan; Yan, Chunri; Lee, Il-Seok; Piao, Xuan-Mei; Byun, Young Joon; Jeong, Pildu; Kim, Won Tae; Yun, Seok-Joong; Kim, Wun-Jae

    2016-01-01

    Purpose Topoisomerase-II alpha (TopoIIA ), a DNA gyrase isoform that plays an important role in the cell cycle, is present in normal tissues and various human cancers, and can show altered expression in both. The aim of the current study was to examine the value of urinary TopoIIA cell-free DNA as a noninvasive diagnosis of bladder cancer (BC). Materials and Methods Two patient cohorts were examined. Cohort 1 (73 BC patients and seven controls) provided bladder tissue samples, whereas cohort ...

  11. Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood

    OpenAIRE

    Fan, H. Christina; Blumenfeld, Yair J.; Chitkara, Usha; Hudgins, Louanne; Quake, Stephen R.

    2008-01-01

    We directly sequenced cell-free DNA with high-throughput shotgun sequencing technology from plasma of pregnant women, obtaining, on average, 5 million sequence tags per patient sample. This enabled us to measure the over- and underrepresentation of chromosomes from an aneuploid fetus. The sequencing approach is polymorphism-independent and therefore universally applicable for the noninvasive detection of fetal aneuploidy. Using this method, we successfully identified all nine cases of trisomy...

  12. Chimeric External Control to Quantify Cell Free DNA in Plasma Samples by Real Time PCR

    Science.gov (United States)

    Eini, Maryam; Behzad-Behbahani, Abbas; Takhshid, Mohammad Ali; Ramezani, Amin; Rafiei Dehbidi, Gholam Reza; Okhovat, Mohammad Ali; Farhadi, Ali; Alavi, Parniyan

    2016-01-01

    Background: DNA isolation procedure can significantly influence the quantification of DNA by real time PCR specially when cell free DNA (cfDNA) is the subject. To assess the extraction efficiency, linearity of the extraction yield, presence of co-purified inhibitors and to avoid problems with fragment size relevant to cfDNA, development of appropriate External DNA Control (EDC) is challenging. Using non-human chimeric nucleotide sequences, an EDC was developed for standardization of qPCR for monitoring stability of cfDNA concentration in blood samples over time. Methods: A0 DNA fragment of 167 bp chimeric sequence of parvovirus B19 and pBHA designated as EDC fragment was designed. To determine the impact of different factors during DNA extraction processing on quantification of cfDNA, blood samples were collected from normal subjects and divided into aliquots with and without specific treatment. In time intervals, the plasma samples were isolated. The amplicon of 167 bp EDC fragment in final concentration of 1.1 pg/500 μl was added to each plasma sample and total DNA was extracted by an in house method. Relative and absolute quantification real time PCR was performed to quantify both EDC fragment and cfDNA in extracted samples. Results: Comparison of real time PCR threshold cycle (Ct) for cfDNA fragment in tubes with and without specific treatment indicated a decrease in untreated tubes. In contrast, the threshold cycle was constant for EDC fragment in treated and untreated tubes, indicating the difference in Ct values of the cfDNA is because of specific treatments that were made on them. Conclusions: Spiking of DNA fragment size relevant to cfDNA into the plasma sample can be useful to minimize the bias due to sample preparation and extraction processing. Therefore, it is highly recommended that standard external DNA control be employed for the extraction and quantification of cfDNA for accurate data analysis.

  13. Methylation of cell-free circulating DNA in the diagnosis of cancer

    Science.gov (United States)

    Warton, Kristina; Samimi, Goli

    2015-01-01

    A range of molecular alterations found in tumor cells, such as DNA mutations and DNA methylation, is reflected in cell-free circulating DNA (circDNA) released from the tumor into the blood, thereby making circDNA an ideal candidate for the basis of a blood-based cancer diagnosis test. In many cancer types, mutations driving tumor development and progression are present in a wide range of oncogenes and tumor suppressor genes. However, even when a gene is consistently mutated in a particular cancer, the mutations can be spread over very large regions of its sequence, making evaluation difficult. This diversity of sequence changes in tumor DNA presents a challenge for the development of blood tests based on DNA mutations for cancer diagnosis. Unlike mutations, DNA methylation that can be consistently measured, as it tends to occur in specific regions of the DNA called CpG islands. Since DNA methylation is reflected within circDNA, detection of tumor-specific DNA methylation in patient plasma is a feasible approach for the development of a blood-based test. Aberrant circDNA methylation has been described in most cancer types and is actively being investigated for clinical applications. A commercial blood test for colorectal cancer based on the methylation of the SEPT9 promoter region in circDNA is under review for approval by the Federal Drug Administration (FDA) for clinical use. In this paper, we review the state of research in circDNA methylation as an application for blood-based diagnostic tests in colorectal, breast, lung, pancreatic and ovarian cancers, and we consider some of the future directions and challenges in this field. There are a number of potential circDNA biomarkers currently under investigation, and experience with SEPT9 shows that the time to clinical translation can be relatively rapid, supporting the promise of circDNA as a biomarker. PMID:25988180

  14. DNA Microgels as a Platform for Cell-Free Protein Expression and Display.

    Science.gov (United States)

    Kahn, Jason S; Ruiz, Roanna C H; Sureka, Swati; Peng, Songming; Derrien, Thomas L; An, Duo; Luo, Dan

    2016-06-13

    Protein expression and selection is an essential process in the modification of biological products. Expressed proteins are selected based on desired traits (phenotypes) from diverse gene libraries (genotypes), whose size may be limited due to the difficulties inherent in diverse cell preparation. In addition, not all genes can be expressed in cells, and linking genotype with phenotype further presents a great challenge in protein engineering. We present a DNA gel-based platform that demonstrates the versatility of two DNA microgel formats to address fundamental challenges of protein engineering, including high protein yield, isolation of gene sets, and protein display. We utilize microgels to show successful protein production and capture of a model protein, green fluorescent protein (GFP), which is further used to demonstrate a successful gene enrichment through fluorescence-activated cell sorting (FACS) of a mixed population of microgels containing the GFP gene. Through psoralen cross-linking of the hydrogels, we have synthesized DNA microgels capable of surviving denaturing conditions while still possessing the ability to produce protein. Lastly, we demonstrate a method of producing extremely high local gene concentrations of up to 32 000 gene repeats in hydrogels 1 to 2 μm in diameter. These DNA gels can serve as a novel cell-free platform for integrated protein expression and display, which can be applied toward more powerful, scalable protein engineering and cell-free synthetic biology with no physiological boundaries and limitations. PMID:27112709

  15. Characterization of the cell-free DNA released by cultured cancer cells.

    Science.gov (United States)

    Bronkhorst, Abel Jacobus; Wentzel, Johannes F; Aucamp, Janine; van Dyk, Etresia; du Plessis, Lissinda; Pretorius, Piet J

    2016-01-01

    The most prominent factor that delays the translation of cell-free DNA (cfDNA) analyses to clinical practice is the lack of knowledge regarding its origin and composition. The elucidation of the former is complicated by the seemingly random fluctuation of quantitative and qualitative characteristics of cfDNA in the blood of healthy and diseased individuals. Besides methodological discrepancies, this could be ascribed to a web of cellular responses to various environmental cues and stressors. Since all cells release cfDNA, it follows that the cfDNA in the blood of cancer patients is not only representative of tumor derived DNA, but also of DNA released by healthy cells under different conditions. Additionally, cfDNA released by malignant cells is not necessarily just aberrant, but likely includes non-mutated chromosomal DNA fragments. This may cause false positive/negative results. Although many have acknowledged that this is a major problem, few have addressed it. We propose that many of the current stumbling blocks encountered in in vivo cfDNA studies can be partially circumvented by in vitro models. Accordingly, the purpose of this work was to evaluate the release of cfDNA from cultured cells and to gauge its potential use for elucidating the nature of cfDNA. Results suggest that the occurrence of cfDNA is not a consequence of apoptosis or necrosis, but primarily a result of actively secreted DNA, perhaps in association with a protein complex. This study demonstrates the potential of in vitro cell culture models to obtain useful information about the phenomenon of cfDNA. PMID:26529550

  16. Levels of plasma circulating cell free nuclear and mitochondrial DNA as potential biomarkers for breast tumors

    Directory of Open Access Journals (Sweden)

    Diesch Claude

    2009-11-01

    Full Text Available Abstract Background With the aim to simplify cancer management, cancer research lately dedicated itself more and more to discover and develop non-invasive biomarkers. In this connection, circulating cell-free DNA (ccf DNA seems to be a promising candidate. Altered levels of ccf nuclear DNA (nDNA and mitochondrial DNA (mtDNA have been found in several cancer types and might have a diagnostic value. Methods Using multiplex real-time PCR we investigated the levels of ccf nDNA and mtDNA in plasma samples from patients with malignant and benign breast tumors, and from healthy controls. To evaluate the applicability of plasma ccf nDNA and mtDNA as a biomarker for distinguishing between the three study-groups we performed ROC (Receiver Operating Characteristic curve analysis. We also compared the levels of both species in the cancer group with clinicopathological parameters. Results While the levels of ccf nDNA in the cancer group were significantly higher in comparison with the benign tumor group (P P P P = 0.022. The level of ccf nDNA was also associated with tumor-size (2 cmP = 0.034. Using ROC curve analysis, we were able to distinguish between the breast cancer cases and the healthy controls using ccf nDNA as marker (cut-off: 1866 GE/ml; sensitivity: 81%; specificity: 69%; P P Conclusion Our data suggests that nuclear and mitochondrial ccf DNA have potential as biomarkers in breast tumor management. However, ccf nDNA shows greater promise regarding sensitivity and specificity.

  17. A cell-free system for DNA repair synthesis using purified enzymes from the Novikoff hepatoma

    International Nuclear Information System (INIS)

    Novikoff DNA polymerase-β and Novikoff DNase V have been used in a cell-free DNA excision repair system for UV-irradiated substrates to determine their DNA repair capabilities. The repair system was shown to depend upon UV-irradiated DNA, incision by phage T4 UV-endonuclease, excision by DNase V and synthesis by DNA polymerase-β; ligation was not included. Highly purified calf thymus DNA was UV-irradiated at 500-750 J/m2 and incised by T4 UV-endonuclease. The repair system was used to follow the purification of DNase V and DNA polymerase-β. For increased specificity, the parameters of UV-irradiation, incision, excision and synthesis were confirmed on highly supercoiled, covalently closed, phage PM2 DNA. Optimal DNA and Mg2+ concentrations were determined for the repair assay, which was shown to be linear with respect to time. Excision of the 3'-apyrimidinic site and the 5'-pyrimidine dimer by bidirectional DNase V, presumed to occur from the above experiments, was studied more thoroughly using lightly UV-irradiated [3H]poly(dT)poly (dA), labeled in both the base and the sugar, and incised with T4 UV-endonuclease

  18. Urine Cell-Free DNA Integrity Analysis for Early Detection of Prostate Cancer Patients

    Science.gov (United States)

    Salvi, Samanta; Gurioli, Giorgia; Martignano, Filippo; Foca, Flavia; Gunelli, Roberta; Cicchetti, Giacomo; De Giorgi, Ugo; Zoli, Wainer; Calistri, Daniele; Casadio, Valentina

    2015-01-01

    Introduction. The detection of tumor-specific markers in urine has paved the way for new early noninvasive diagnostic approaches for prostate cancer. We evaluated the DNA integrity in urine supernatant to verify its capacity to discriminate between prostate cancer and benign diseases of the urogenital tract. Patients and Methods. A total of 131 individuals were enrolled: 67 prostate cancer patients and 64 patients with benign diseases of the urogenital tract (control group). Prostate-specific antigen (PSA) levels were determined. Urine cell-free (UCF) DNA was isolated and sequences longer than 250 bp corresponding to 3 genes (c-MYC, HER2, and AR) were quantified by Real-Time PCR to assess UCF-DNA integrity. Results. UCF-DNA was quantifiable in all samples, while UCF-DNA integrity was evaluable in all but 16 samples. Receiver operating characteristic analysis showed an area under the curve of 0.5048 for UCF-DNA integrity and 0.8423 for PSA. Sensitivity was 0.58 and 0.95 for UCF-DNA integrity and PSA, respectively. Specificity was 0.44 and 0.69, respectively. Conclusions. UCF-DNA integrity showed lower accuracy than PSA and would not seem to be a reliable marker for early prostate cancer diagnosis. Despite this, we believe that UCF-DNA could represent a source of other biomarkers and could detect gene alterations. PMID:26412928

  19. Cell-free DNA next-generation sequencing in pancreatobiliary carcinomas

    Science.gov (United States)

    Zill, Oliver A.; Greene, Claire; Sebisanovic, Dragan; Siew, LaiMun; Leng, Jim; Vu, Mary; Hendifar, Andrew E.; Wang, Zhen; Atreya, Chloe E.; Kelley, Robin K.; Van Loon, Katherine; Ko, Andrew H.; Tempero, Margaret A.; Bivona, Trever G.; Munster, Pamela N.; Talasaz, AmirAli; Collisson, Eric A.

    2015-01-01

    Patients with pancreatic and biliary carcinomas lack personalized treatment options, in part because biopsies are often inadequate for molecular characterization. Cell-free DNA (cfDNA) sequencing may enable a precision oncology approach in this setting. We attempted to prospectively analyze 54 genes in tumor and cfDNA for 26 patients. Tumor sequencing failed in nine patients (35%). In the remaining 17, 90.3% (95% CI: 73.1–97.5%) of mutations detected in tumor biopsies were also detected in cfDNA. The diagnostic accuracy of cfDNA sequencing was 97.7%, with 92.3% average sensitivity and 100% specificity across five informative genes. Changes in cfDNA correlated well with tumor marker dynamics in serial sampling (r=0.93). We demonstrate that cfDNA sequencing is feasible, accurate, and sensitive in identifying tumor-derived mutations without prior knowledge of tumor genotype or the abundance of circulating tumor DNA. cfDNA sequencing should be considered in pancreatobiliary cancer trials where tissue sampling is unsafe, infeasible, or otherwise unsuccessful. PMID:26109333

  20. Urine Cell-Free DNA Integrity Analysis for Early Detection of Prostate Cancer Patients

    Directory of Open Access Journals (Sweden)

    Samanta Salvi

    2015-01-01

    Full Text Available Introduction. The detection of tumor-specific markers in urine has paved the way for new early noninvasive diagnostic approaches for prostate cancer. We evaluated the DNA integrity in urine supernatant to verify its capacity to discriminate between prostate cancer and benign diseases of the urogenital tract. Patients and Methods. A total of 131 individuals were enrolled: 67 prostate cancer patients and 64 patients with benign diseases of the urogenital tract (control group. Prostate-specific antigen (PSA levels were determined. Urine cell-free (UCF DNA was isolated and sequences longer than 250 bp corresponding to 3 genes (c-MYC, HER2, and AR were quantified by Real-Time PCR to assess UCF-DNA integrity. Results. UCF-DNA was quantifiable in all samples, while UCF-DNA integrity was evaluable in all but 16 samples. Receiver operating characteristic analysis showed an area under the curve of 0.5048 for UCF-DNA integrity and 0.8423 for PSA. Sensitivity was 0.58 and 0.95 for UCF-DNA integrity and PSA, respectively. Specificity was 0.44 and 0.69, respectively. Conclusions. UCF-DNA integrity showed lower accuracy than PSA and would not seem to be a reliable marker for early prostate cancer diagnosis. Despite this, we believe that UCF-DNA could represent a source of other biomarkers and could detect gene alterations.

  1. Direct quantification of cell-free, circulating DNA from unpurified plasma.

    Directory of Open Access Journals (Sweden)

    Sarah Breitbach

    Full Text Available Cell-free DNA (cfDNA in body tissues or fluids is extensively investigated in clinical medicine and other research fields. In this article we provide a direct quantitative real-time PCR (qPCR as a sensitive tool for the measurement of cfDNA from plasma without previous DNA extraction, which is known to be accompanied by a reduction of DNA yield. The primer sets were designed to amplify a 90 and 222 bp multi-locus L1PA2 sequence. In the first module, cfDNA concentrations in unpurified plasma were compared to cfDNA concentrations in the eluate and the flow-through of the QIAamp DNA Blood Mini Kit and in the eluate of a phenol-chloroform isoamyl (PCI based DNA extraction, to elucidate the DNA losses during extraction. The analyses revealed 2.79-fold higher cfDNA concentrations in unpurified plasma compared to the eluate of the QIAamp DNA Blood Mini Kit, while 36.7% of the total cfDNA were found in the flow-through. The PCI procedure only performed well on samples with high cfDNA concentrations, showing 87.4% of the concentrations measured in plasma. The DNA integrity strongly depended on the sample treatment. Further qualitative analyses indicated differing fractions of cfDNA fragment lengths in the eluate of both extraction methods. In the second module, cfDNA concentrations in the plasma of 74 coronary heart disease patients were compared to cfDNA concentrations of 74 healthy controls, using the direct L1PA2 qPCR for cfDNA quantification. The patient collective showed significantly higher cfDNA levels (mean (SD 20.1 (23.8 ng/ml; range 5.1-183.0 ng/ml compared to the healthy controls (9.7 (4.2 ng/ml; range 1.6-23.7 ng/ml. With our direct qPCR, we recommend a simple, economic and sensitive procedure for the quantification of cfDNA concentrations from plasma that might find broad applicability, if cfDNA became an established marker in the assessment of pathophysiological conditions.

  2. Circulating Cell-Free Tumour DNA in the Management of Cancer

    Science.gov (United States)

    Francis, Glenn; Stein, Sandra

    2015-01-01

    With the development of new sensitive molecular techniques, circulating cell-free tumour DNA containing mutations can be identified in the plasma of cancer patients. The applications of this technology may result in significant changes to the care and management of cancer patients. Whilst, currently, these “liquid biopsies” are used to supplement the histological diagnosis of cancer and metastatic disease, in the future these assays may replace the need for invasive procedures. Applications include the monitoring of tumour burden, the monitoring of minimal residual disease, monitoring of tumour heterogeneity, monitoring of molecular resistance and early diagnosis of tumours and metastatic disease. PMID:26101870

  3. Circulating Cell-Free Tumour DNA in the Management of Cancer

    Directory of Open Access Journals (Sweden)

    Glenn Francis

    2015-06-01

    Full Text Available With the development of new sensitive molecular techniques, circulating cell-free tumour DNA containing mutations can be identified in the plasma of cancer patients. The applications of this technology may result in significant changes to the care and management of cancer patients. Whilst, currently, these “liquid biopsies” are used to supplement the histological diagnosis of cancer and metastatic disease, in the future these assays may replace the need for invasive procedures. Applications include the monitoring of tumour burden, the monitoring of minimal residual disease, monitoring of tumour heterogeneity, monitoring of molecular resistance and early diagnosis of tumours and metastatic disease.

  4. Feasibility of cell-free circulating tumor DNA testing for lung cancer.

    Science.gov (United States)

    Santarpia, Mariacarmela; Karachaliou, Niki; González-Cao, Maria; Altavilla, Giuseppe; Giovannetti, Elisa; Rosell, Rafael

    2016-04-01

    Tumor tissue genotyping is used routinely for lung cancer to identify specific targetable oncogenic alterations, including EGFR mutations and ALK rearrangements. However, tumor tissue from a single biopsy is often insufficient for molecular testing, may offer a limited evaluation because of tumor heterogeneity and can be difficult to obtain. Cell-free circulating tumor DNA has been widely investigated as a potential surrogate for tissue biopsy for noninvasive assessment of tumor-related genomic alterations. New techniques have improved EGFR mutations detection in ctDNA, thus supporting the use of this liquid biopsy for predicting response to EGFR tyrosine kinase inhibitors (TKIs) and monitoring the emergence of resistance. The serial evaluation of ctDNA during treatment is feasible and can be used to track tumor changes in real time and for a wide range of clinically useful applications. PMID:26974841

  5. The Prognostic Value of Circulating Cell-Free DNA in Colorectal Cancer: A Meta-Analysis

    Science.gov (United States)

    Basnet, Shiva; Zhang, Zhen-yu; Liao, Wen-qiang; Li, Shu-heng; Li, Ping-shu; Ge, Hai-yan

    2016-01-01

    Background: Circulating cell-free DNA (cfDNA) is a promising candidate biomarker for detection, monitoring and survival prediction of colorectal cancer (CRC). However, its prognostic significance for patients with CRC remains controversial. To derive a precise estimation of the prognostic significance of cfDNA, a meta-analysis was performed. Methods: We made a systematic search in data base of the Science Citation Index Embase and Pubmed for studies reporting prognostic data of cfDNA in CRC patients. The data of cfDNA on recurrences-free survival (RFS) and overall survival (OS) were extracted and measured in hazard rates (HRs) and 95% confident intervals (CIs). Subgroup analyses were carried out as well. Finally, the meta-analysis is accompanied with nine studies including 19 subunits. Results: The pooled HRs with 95% CIs revealed strong associations between cfDNA and RFS (HR [95%CI]=2.78[2.08-3.72], I2=32.23%, n=7) along with OS (HR [95%CI]=3.03[2.51-3.66], I2=29.24%, n=12) in patients with CRC. Entire subgroup analyses indicated strong prognostic value of cfDNA irrespective tumor stage, study size, tumor markers, detection methods and marker origin. Conclusions: All the results exhibits that appearance of cfDNA in blood is an indicator for adverse RFS and OS in CRC patients. PMID:27326254

  6. Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma.

    Science.gov (United States)

    Burnham, Philip; Kim, Min Seong; Agbor-Enoh, Sean; Luikart, Helen; Valantine, Hannah A; Khush, Kiran K; De Vlaminck, Iwijn

    2016-01-01

    Circulating cell-free DNA (cfDNA) is emerging as a powerful monitoring tool in cancer, pregnancy and organ transplantation. Nucleosomal DNA, the predominant form of plasma cfDNA, can be adapted for sequencing via ligation of double-stranded DNA (dsDNA) adapters. dsDNA library preparations, however, are insensitive to ultrashort, degraded cfDNA. Drawing inspiration from advances in paleogenomics, we have applied a single-stranded DNA (ssDNA) library preparation method to sequencing of cfDNA in the plasma of lung transplant recipients (40 samples, six patients). We found that ssDNA library preparation yields a greater portion of sub-100 bp nuclear genomic cfDNA (p 10(-5), Mann-Whitney U Test), and an increased relative abundance of mitochondrial (10.7x, p 10(-5)) and microbial cfDNA (71.3x, p 10(-5)). The higher yield of microbial sequences from this method increases the sensitivity of cfDNA-based monitoring for infections following transplantation. We detail the fragmentation pattern of mitochondrial, nuclear genomic and microbial cfDNA over a broad fragment length range. We report the observation of donor-specific mitochondrial cfDNA in the circulation of lung transplant recipients. A ssDNA library preparation method provides a more informative window into understudied forms of cfDNA, including mitochondrial and microbial derived cfDNA and short nuclear genomic cfDNA, while retaining information provided by standard dsDNA library preparation methods. PMID:27297799

  7. Plasma cell-free mitochondrial DNA declines in response to prolonged moderate aerobic exercise.

    Science.gov (United States)

    Shockett, Penny E; Khanal, Januka; Sitaula, Alina; Oglesby, Christopher; Meachum, William A; Castracane, V Daniel; Kraemer, Robert R

    2016-01-01

    Increased plasma cell-free mitochondrial DNA (cf-mDNA), a damage-associated molecular pattern (DAMP) produced by cellular injury, contributes to neutrophil activation/inflammation in trauma patients and arises in cancer and autoimmunity. To further understand relationships between cf-mDNA released by tissue injury, inflammation, and health benefits of exercise, we examined cf-mDNA response to prolonged moderate aerobic exercise. Seven healthy moderately trained young men (age = 22.4 ± 1.2) completed a treadmill exercise trial for 90 min at 60% VO2 max and a resting control trial. Blood was sampled immediately prior to exercise (0 min = baseline), during (+18, +54 min), immediately after (+90 min), and after recovery (R40). Plasma was analyzed for cf-mDNA, IL-6, and lactate. A significant difference in cf-mDNA response was observed between exercise and control trials, with cf-mDNA levels reduced during exercise at +54 and +90 (with or without plasma volume shift correction). Declines in cf-mDNA were accompanied by increased lactate and followed by an increase in IL-6, suggesting a temporal association with muscle stress and inflammatory processes. Our novel finding of cf-mDNA decline with prolonged moderate treadmill exercise provides evidence for increased clearance from or reduced release of cf-mDNA into the blood with prolonged exercise. These studies contrast with previous investigations involving exhaustive short-term treadmill exercise, in which no change in cf-mDNA levels were reported, and contribute to our understanding of differences between exercise- and trauma-induced inflammation. We propose that transient declines in cf-mDNA may induce health benefits, by reducing systemic inflammation. PMID:26755735

  8. Plasma cell-free DNA levels are elevated in acute Puumala hantavirus infection.

    Directory of Open Access Journals (Sweden)

    Tuula K Outinen

    Full Text Available INTRODUCTION: Puumala hantavirus (PUUV causes a hemorrhagic fever with renal syndrome called nephropathia epidemica (NE. The aim of the present study was to evaluate plasma cell-free DNA (cf-DNA levels and urinary cf-DNA excretion in acute NE as well as their associations with the severity of the disease. METHODS: Total plasma cf-DNA was quantified directly in plasma of 61 patients and urine of 20 patients with acute NE. We also carried out a qualitative high-sensitivity lab-on-a-chip DNA assay in 20 patients to elucidate the appearance of cf-DNA in plasma and urine. RESULTS: The maximum plasma cf-DNA values taken during acute NE were significantly higher than the control values taken after the hospitalization period (median 1.33 µg/ml, range 0.94-3.29 µg/ml vs. median 0.77 µg/ml, range 0.55-0.99 µg/ml, P<0.001. The maximum plasma cf-DNA levels correlated positively with maximum blood leukocyte count (r = 0.388, P = 0.002 and the length of hospital stay (r = 0.376, P = 0.003, and inversely with minimum blood platelet count (r = -0.297, P = 0.020. Qualitative analysis of plasma cf-DNA revealed that in most of the patients cf-DNA displayed a low-molecular weight appearance, corresponding to the size of apoptotic DNA (150-200 bp. The visually graded maximum cf-DNA band intensity correlated positively with the maximum quantity of total plasma cf-DNA (r = 0.513, P = 0.021. Maximum urinary excretion of cf-DNA in turn was not markedly increased during the acute phase of NE and did not correlate with any of the variables reflecting severity of the disease or with the maximum plasma cf-DNA level. CONCLUSIONS: The plasma levels of cf-DNA are elevated during acute PUUV infection and correlate with the apoptotic cf-DNA-band intensity. The plasma cf-DNA concentration correlates with some variables reflecting the severity of the disease. The urinary excretion of cf-DNA does not reflect the degree of inflammation in the kidney.

  9. Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood.

    Science.gov (United States)

    Fan, H Christina; Blumenfeld, Yair J; Chitkara, Usha; Hudgins, Louanne; Quake, Stephen R

    2008-10-21

    We directly sequenced cell-free DNA with high-throughput shotgun sequencing technology from plasma of pregnant women, obtaining, on average, 5 million sequence tags per patient sample. This enabled us to measure the over- and underrepresentation of chromosomes from an aneuploid fetus. The sequencing approach is polymorphism-independent and therefore universally applicable for the noninvasive detection of fetal aneuploidy. Using this method, we successfully identified all nine cases of trisomy 21 (Down syndrome), two cases of trisomy 18 (Edward syndrome), and one case of trisomy 13 (Patau syndrome) in a cohort of 18 normal and aneuploid pregnancies; trisomy was detected at gestational ages as early as the 14th week. Direct sequencing also allowed us to study the characteristics of cell-free plasma DNA, and we found evidence that this DNA is enriched for sequences from nucleosomes. PMID:18838674

  10. Plasma circulating cell-free mitochondrial DNA in the assessment of Friedreich's ataxia.

    Science.gov (United States)

    Dantham, Subrahamanyam; Srivastava, Achal K; Gulati, Sheffali; Rajeswari, Moganty R

    2016-06-15

    Friedreich's ataxia (FRDA) is one of the most devastating childhood onset neurodegenerative disease affecting multiple organs in the course of progression. FRDA is associated with mitochondrial dysfunction due to deficit in a nuclear encoded mitochondrial protein, frataxin. Identification of disease-specific biomarker for monitoring the severity remains to be a challenging topic. This study was aimed to identify whether circulating cell-free nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) in blood plasma can be a potential biomarker for FRDA. Clinical information was assessed using International Cooperative Ataxia Rating Scale and the disease was confirmed using Long-range PCR for GAA repeat expansion within the gene encoding frataxin. The frataxin expression was measured using Western blot. Plasma nDNA and mtDNA levels were quantified by Multiplex real-time PCR. The major observation is that the levels of nDNA found to be increased, whereas mtDNA levels were reduced significantly in the plasma of FRDA patients (n=21) as compared to healthy controls (n=21). Further, plasma mtDNA levels showed high sensitivity (90%) and specificity (76%) in distinguishing from healthy controls with optimal cutoff indicated at 4.1×10(5)GE/mL. Interestingly, a small group of follow-up patients (n=9) on intervention with, a nutrient supplement, omega-3 fatty acid (a known enhancer of mitochondrial metabolism) displayed a significant improvement in the levels of plasma mtDNA, supporting our hypothesis that plasma mtDNA can be a potential monitoring or prognosis biomarker for FRDA. PMID:27206881

  11. TUMOR-RELATED METHYLATED CELL-FREE DNA AND CIRCULATING TUMOR CELLS IN MELANOMA

    Directory of Open Access Journals (Sweden)

    Francesca eSalvianti

    2016-01-01

    Full Text Available Solid tumor release into the circulation cell-free DNA (cfDNA and circulating tumor cells (CTCs which represent promising biomarkers for cancer diagnosis. Circulating tumor DNA may be studied in plasma from cancer patients by detecting tumor specific alterations, such as genetic or epigenetic modifications. Ras association domain family 1 isoform A (RASSF1A is a tumor suppressor gene silenced by promoter hypermethylation in a variety of human cancers including melanoma.The aim of the present study was to assess the diagnostic performance of a tumor-related methylated cfDNA marker in melanoma patients and to compare this parameter with the presence of CTCs.RASSF1A promoter methylation was quantified in cfDNA by qPCR in a consecutive series of 84 melanoma patients and 68 healthy controls. In a subset of 68 cases, the presence of CTCs was assessed by a filtration method (Isolation by Size of Epithelial Tumor Cells, ISET as well as by an indirect method based on the detection of tyrosinase mRNA by RT-qPCR. The distribution of RASSF1A methylated cfDNA was investigated in cases and controls and the predictive capability of this parameter was assessed by means of the area under the ROC curve (AUC.The percentage of cases with methylated RASSF1A promoter in cfDNA was significantly higher in each class of melanoma patients (in situ, invasive and metastatic than in healthy subjects (Pearson chi-squared test, p<0.001. The concentration of RASSF1A methylated cfDNA in the subjects with a detectable quantity of methylated alleles was significantly higher in melanoma patients than in controls. The biomarker showed a good predictive capability (in terms of AUC in discriminating between melanoma patients and healthy controls. This epigenetic marker associated to cfDNA did not show a significant correlation with the presence of CTCs, but, when the two parameters are jointly considered, we obtain a higher sensitivity of the detection of positive cases in invasive

  12. Tumor-Related Methylated Cell-Free DNA and Circulating Tumor Cells in Melanoma

    Science.gov (United States)

    Salvianti, Francesca; Orlando, Claudio; Massi, Daniela; De Giorgi, Vincenzo; Grazzini, Marta; Pazzagli, Mario; Pinzani, Pamela

    2016-01-01

    Solid tumor release into the circulation cell-free DNA (cfDNA) and circulating tumor cells (CTCs) which represent promising biomarkers for cancer diagnosis. Circulating tumor DNA may be studied in plasma from cancer patients by detecting tumor specific alterations, such as genetic or epigenetic modifications. Ras association domain family 1 isoform A (RASSF1A) is a tumor suppressor gene silenced by promoter hypermethylation in a variety of human cancers including melanoma. The aim of the present study was to assess the diagnostic performance of a tumor-related methylated cfDNA marker in melanoma patients and to compare this parameter with the presence of CTCs. RASSF1A promoter methylation was quantified in cfDNA by qPCR in a consecutive series of 84 melanoma patients and 68 healthy controls. In a subset of 68 cases, the presence of CTCs was assessed by a filtration method (Isolation by Size of Epithelial Tumor Cells, ISET) as well as by an indirect method based on the detection of tyrosinase mRNA by RT-qPCR. The distribution of RASSF1A methylated cfDNA was investigated in cases and controls and the predictive capability of this parameter was assessed by means of the area under the ROC curve (AUC). The percentage of cases with methylated RASSF1A promoter in cfDNA was significantly higher in each class of melanoma patients (in situ, invasive and metastatic) than in healthy subjects (Pearson chi-squared test, p < 0.001). The concentration of RASSF1A methylated cfDNA in the subjects with a detectable quantity of methylated alleles was significantly higher in melanoma patients than in controls. The biomarker showed a good predictive capability (in terms of AUC) in discriminating between melanoma patients and healthy controls. This epigenetic marker associated to cfDNA did not show a significant correlation with the presence of CTCs, but, when the two parameters are jointly considered, we obtain a higher sensitivity of the detection of positive cases in invasive and

  13. Nucleotide-excision repair of DNA in cell-free extracts of the yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    A wide spectrum of DNA lesions are repaired by the nucleotide-excision repair (NER) pathway in both eukaryotic and prokaryotic cells. We have developed a cell-free system in Saccharomyces cerevisiae that supports NER. NER was monitored by measuring repair synthesis in DNA treated with cisplatin or with UV radiation. Repair synthesis in vitro was defective in extracts of rad1, rad2, and rad10 mutant cells, all of which have mutations in genes whose products are known to be required for NER in vivo. Additionally, repair synthesis was complemented by mixing different mutant extracts, or by adding purified Rad1 or Rad10 protein to rad1 or rad10 mutant extracts, respectively. The latter observation demonstrates that the Rad1 and Rad10 proteins directly participate in the biochemical pathway of NER. NER supported by nuclear extracts requires ATP and Mg2+ and is stimulated by polyethylene glycol and by small amounts of whole cell extract containing overexpressed Rad2 protein. The nuclear extracts also contain base-excision repair activity that is present at wild-type levels in rad mutant extracts. This cell-free system is expected to facilitate studies on the biochemical pathway of NER in S. cerevisiae

  14. Methylation of cell-free circulating DNA in the diagnosis of cancer

    Directory of Open Access Journals (Sweden)

    Goli eSamimi

    2015-04-01

    Full Text Available A range of molecular alterations found in tumor cells, such as DNA mutations and methylation changes, is also reflected in cell-free circulating DNA (circDNA released from the tumor into the blood, thereby making circDNA an ideal candidate for the basis of a blood-based cancer diagnosis test. In many cancer types, mutations driving tumor development and progression are present in a wide range of oncogenes and tumor suppressor genes. However, even when a gene is consistently mutated in a particular cancer, the mutations can be spread over very large regions of its sequence, making evaluation difficult. This diversity of sequence changes in tumor DNA presents a challenge for the development of blood tests based on DNA mutations for cancer diagnosis. DNA methylation is a common molecular alteration found in many cancer types. Unlike DNA mutations, DNA methylation that can be consistently measured, as it tends to occur in specific regions of the DNA called CpG islands. DNA methylation is reflected within circDNA and therefore detection of tumor-specific DNA methylation in patient plasma is a feasible approach for the development of a blood-based test. Aberrant circDNA methylation has been described in most cancer types and is actively being investigated for clinical applications. A commercial blood test for colorectal cancer based on the methylation of the SEPT9 promoter region in circDNA is under review for approval by the Federal Drug Administration (FDA for clinical use. In this paper, we review the state of research in circDNA methylation as an application for blood-based diagnostic tests in colorectal, breast, lung, pancreatic and ovarian cancers, and we consider some of the future directions and challenges in this field. There are a number of potential circDNA biomarkers currently under investigation, and experience with SEPT9 shows that the time to clinical translation can be relatively rapid, supporting the promise of circDNA as a biomarker.

  15. Maternal plasma fetal DNA fractions in pregnancies with low and high risks for fetal chromosomal aneuploidies.

    Directory of Open Access Journals (Sweden)

    Irena Hudecova

    Full Text Available Recently published international guidelines recommend the clinical use of noninvasive prenatal test (NIPT for aneuploidy screening only among pregnant women whose fetuses are deemed at high risk. The applicability of NIPT to aneuploidy screening among average risk pregnancies requires additional supportive evidence. A key determinant of the reliability of aneuploidy NIPT is the fetal DNA fraction in maternal plasma. In this report, we investigated if differences in fetal DNA fractions existed between different pregnancy risk groups. One hundred and ninety-five singleton pregnancies with male fetuses divided into 3 groups according to first trimester screening parameters were examined for fetal DNA percentage by counting Y chromosome DNA sequences using massively parallel sequencing. Fetal DNA fractions were compared between risk groups and assessed for correlations with first trimester screening parameters. There was no statistically significant difference in fetal DNA fractions across the high, intermediate and low risk groups. Fetal DNA fraction showed a strong negative correlation with maternal weight. Fetal DNA fraction also showed weak but significant correlations with gestational age, crown-rump length, multiple of medians of free β-subunit of human chorionic gonadotropin and pregnancy-associated plasma protein A. Similar fetal DNA fractions in maternal plasma between high, intermediate and low risk pregnant women is a precondition for uniform performance of the aneuploidy NIPTs for the general population. This study thus shows that the aneuploidy screening by NIPT is likely to offer similar analytical reliability without respect to the a priori fetal aneuploidy risk.

  16. Quantitation of cell-free DNA and RNA in plasma during tumor progression in rats

    Directory of Open Access Journals (Sweden)

    García-Olmo Dolores C

    2013-02-01

    Full Text Available Abstract Background To clarify the implications of cell-free nucleic acids (cfNA in the plasma in neoplastic disease, it is necessary to determine the kinetics of their release into the circulation. Methods To quantify non-tumor and tumor DNA and RNA in the plasma of tumor-bearing rats and to correlate such levels with tumor progression, we injected DHD/K12-PROb colon cancer cells subcutaneously into syngenic BD-IX rats. Rats were sacrificed and their plasma was analyzed from the first to the eleventh week after inoculation. Results The release of large amounts of non-tumor DNA into plasma was related to tumor development from its early stages. Tumor-specific DNA was detected in 33% of tumor-bearing rats, starting from the first week after inoculation and at an increasing frequency thereafter. Animals that were positive for tumor DNA in the plasma had larger tumors than those that were negative (p = 0.0006. However, the appearance of both mutated and non-mutated DNA fluctuated with time and levels of both were scattered among individuals in each group. The release of non-tumor mRNA was unaffected by tumor progression and we did not detect mutated RNA sequences in any animals. Conclusions The release of normal and tumor cfDNA into plasma appeared to be related to individual-specific factors. The contribution of tumor DNA to the elevated levels of plasma DNA was intermittent. The release of RNA into plasma during cancer progression appeared to be an even more selective and elusive phenomenon than that of DNA.

  17. Cell-free DNA in healthy individuals, noncancerous disease and strong prognostic value in colorectal cancer

    DEFF Research Database (Denmark)

    Spindler, Karen-Lise Garm; Appelt, Ane L; Pallisgaard, Niels;

    2014-01-01

    The purpose was to investigate total cell-free DNA (cfDNA) in colorectal cancer (CRC) patients during treatment with second-line chemotherapy and in healthy controls and patients with different comorbidities. Patient treated with second-line irinotecan for metastatic CRC (n = 100), a cohort of...... healthy controls with and without comorbidity (n = 70 and 100, respectively) were included. cfDNA was quantified by an in-house developed quantitative polymerase chain reaction from plasma samples drawn prior to the first cycle of chemotherapy and at time of progression. cfDNA levels were significantly.......8 months (95% CI 11.9-18.9; HR 2.52; 95% CI 1.54-4.13, p < 0.0000), respectively. Cox regression multivariate analysis showed a PFS HR of 1.4 (95% CI 1.1-1.7) for each increase in cfDNA quartile, p = 0.03 and 1.6 (1.3-2.0) for OS, p < 0.0001, respectively. A combined marker analysis with plasma KRAS...

  18. SNPase-ARMS qPCR: Ultrasensitive Mutation-Based Detection of Cell-Free Tumor DNA in Melanoma Patients

    OpenAIRE

    Stadler, Julia; Eder, Johanna; Pratscher, Barbara; Brandt, Sabine; Schneller, Doris; Müllegger, Robert; Vogl, Claus; Trautinger, Franz; Brem, Gottfried; Burgstaller, Joerg P.

    2015-01-01

    Cell-free circulating tumor DNA in the plasma of cancer patients has become a common point of interest as indicator of therapy options and treatment response in clinical cancer research. Especially patient- and tumor-specific single nucleotide variants that accurately distinguish tumor DNA from wild type DNA are promising targets. The reliable detection and quantification of these single-base DNA variants is technically challenging. Currently, a variety of techniques is applied, with no appar...

  19. Cell Free DNA of Tumor Origin Induces a ‘Metastatic’ Expression Profile in HT-29 Cancer Cell Line

    OpenAIRE

    Fűri, István; Kalmár, Alexandra; Wichmann, Barnabás; Spisák, Sándor; Schöller, Andrea; Barták, Barbara; Tulassay, Zsolt; Molnár, Béla

    2015-01-01

    Background Epithelial cells in malignant conditions release DNA into the extracellular compartment. Cell free DNA of tumor origin may act as a ligand of DNA sensing mechanisms and mediate changes in epithelial-stromal interactions. Aims To evaluate and compare the potential autocrine and paracrine regulatory effect of normal and malignant epithelial cell-related DNA on TLR9 and STING mediated pathways in HT-29 human colorectal adenocarcinoma cells and normal fibroblasts. Materials and Methods...

  20. Circulating cell-free DNA indicates M1/M2 responses during septic peritonitis.

    Science.gov (United States)

    Xin, Yi; Gao, Xingjuan; Wang, Wenxiao; Xu, Xiaojuan; Yu, Lijuan; Ju, Xiuli; Li, Aimin

    2016-09-01

    Circulating cell-free DNA (cfDNA) has been widely suggested as clinical indicator in diseases, including sepsis. It was thought that the cfDNA was coming from the cell lysis, necrosis and apoptosis caused by tissue damages during sepsis. M1 or M2 macrophage-type responses kill or repair in vivo, which is highly relevant with the tissue damages in sepsis. The correlation between cfDNA and M1/M2 responses during sepsis was never investigated. Here, we used bacteria injection induced septic peritonitis mouse model in both M1-dominant C57bl/6 and M2-dominant Balb/c mouse strains. We found that M2-dominant Balb/c mice showed better prognosis of septic peritonitis than C57bl/6 mice, which is corresponded with lower level of cfDNA in septic Balb/c mice compared to septic C57bl/6 mice. By assessing the M1 and M2 related cytokines in both septic Balb/c and C57bl/6 mice, we found out that Balb/c mice has lower tumor necrosis factor α (TNFα) and higher interleukin 10 (IL-10) productions than C57bl/6 mice during septic peritonitis. Especially, when monitoring the monocyte subtypes in peripheral blood of these septic mice, we found out that C57bl/6 showed higher inflammatory (Ly6C(high)) monocyte (corresponding to M1 macrophage) proportion than Balb/c mice. Interestingly, we find out that cfDNA is highly correlated with the ratio of Ly6C(high) monocytes versus Ly6C(low) monocytes, which represents M1/M2 (killing/healing) responses. Our study suggested that the cfDNA is a good indicator for evaluating M1/M2 responses in septic peritonitis. PMID:27335257

  1. Circulating cell-free DNA: an up-coming molecular marker in exercise physiology.

    Science.gov (United States)

    Breitbach, Sarah; Tug, Suzan; Simon, Perikles

    2012-07-01

    The phenomenon of circulating cell-free DNA (cfDNA) concentrations is of importance for many biomedical disciplines including the field of exercise physiology. Increases of cfDNA due to exercise are described to be a potential hallmark for the overtraining syndrome and might be related to, or trigger adaptations of, immune function induced by strenuous exercise. At the same time, exercise provides a practicable model for studying the phenomenon of cfDNA that is described to be of pathophysiological relevance for different topics in clinical medicine like autoimmune diseases and cancer. In this review, we are summarizing the current knowledge of exercise-based acute and chronic alterations in cfDNA levels and their physiological significance. The effects of acute exercise on cfDNA concentrations have been investigated in resistance exercises and in continuous, stepwise and interval endurance exercises of different durations. cfDNA concentrations peaked immediately after acute exercise and showed a rapid return to baseline levels. Typical markers of skeletal muscle damage (creatine kinase, uric acid, C-reactive protein) show delayed kinetics compared with the cfDNA peak response. Exercise parameters such as intensity, duration or average energy expenditure do not explain the extent of increasing cfDNA concentrations after strenuous exercise. This could be due to complex processes inside the human organism during and after physical activity. Therefore, we hypothesize composite effects of different physiological stress parameters that come along with exercise to be responsible for increasing cfDNA concentrations. We suggest that due to acute stress, cfDNA levels increase rapidly by a spontaneous active or passive release mechanism that is not yet known. As a result of the rapid and parallel increase of cfDNA and lactate in an incremental treadmill test leading to exhaustion within 15-20 minutes, it is unlikely that cfDNA is released into the plasma by typical necrosis

  2. Unfair discrimination in prenatal aneuploidy screening using cell-free DNA?

    Science.gov (United States)

    Rolfes, Vasilija; Schmitz, Dagmar

    2016-03-01

    Non-invasive prenatal testing on the basis of cell-free DNA of placental origin (NIPT) changed the landscape of prenatal care and is seen as superior to all other up to now implemented prenatal screening procedures - at least in the high-risk population. NIPT has spread almost worldwide commercially, but only in a few countries the costs of NIPT are covered by insurance companies. Such financial barriers in prenatal testing can lead to significant restrictions to the average range of opportunities of pregnant women and couples, which on an intersubjective level can be defined as unfair discrimination and on an individual level weakens reproductive autonomy. Given that enabling reproductive autonomy is the main ethical justification for offering prenatal (genetic) testing, these barriers are not only an issue of justice in health care, but are potentially counteracting the primary purpose of these testing procedures. PMID:26773245

  3. PIK3CA mutation detection in metastatic biliary cancer using cell-free DNA

    Science.gov (United States)

    Deng, Shibing; Lee, Sujin; Park, Young Suk; Lim, Ho Yeong; Kang, Won Ki; Mao, Mao; Heo, Jin Seok; Kwon, Wooil; Jang, Kee-Taek; Lee, Jeeyun; Park, Joon Oh

    2015-01-01

    PIK3CA mutation is considered a good candidate for targeted therapies in cancers, especially biliary tract cancer (BTC). We evaluated the utility of cell free DNA (cfDNA) from serum by using droplet digital PCR (ddPCR) as an alternative source for PIK3CA mutation analysis. To identify matching archival tumour specimens from serum samples of advanced BTC patients, mutation detection using ddPCR with Bio-Rad's PrimePCR mutation and wild type assays were performed for PIK3CA p.E542K, p.E545K, and p.H1047R. Thirty-eight patients with metastatic BTC were enrolled. Only one (BTC 29T) sample (n = 38) was positive for PIK3CA p.E542K and another (BTC 27T) for p.H1047R mutation; none was positive for PIK3CA p.E545K. Matched serum sample (BTC 29P) was positive for PIK3CA p.E542K with 28 mutant copies detected, corresponding to 48 copies/ml of serum and an allelic prevalence of 0.3%. Another matched serum sample (BTC 27P) was positive for PIK3CA p.H1047R with 10 mutant copies detected, i.e. 18 copies/ml and an allelic frequency of 0.2%. High correlation was noted in the PIK3CA mutation status between tumour gDNA and serum cfDNA. Low-level PIK3CA mutations were detectable in the serum indicating the utility of cfDNA as a DNA source to detect cancer-derived mutations in metastatic biliary cancers. PMID:26498688

  4. Aberrant reduction of telomere repetitive sequences in plasma cell-free DNA for early breast cancer detection

    OpenAIRE

    Wu, Xi; Tanaka, Hiromi

    2015-01-01

    Excessive telomere shortening is observed in breast cancer lesions when compared to adjacent non-cancerous tissues, suggesting that telomere length may represent a key biomarker for early cancer detection. Because tumor-derived, cell-free DNA (cfDNA) is often released from cancer cells and circulates in the bloodstream, we hypothesized that breast cancer development is associated with changes in the amount of telomeric cfDNA that can be detected in the plasma. To test this hypothesis, we devi...

  5. Single step generation of protein arrays from DNA by cell-free expression and in situ immobilisation (PISA method)

    OpenAIRE

    He, Mingyue; Michael J. Taussig

    2001-01-01

    We describe a format for production of protein arrays termed ‘protein in situ array’ (PISA). A PISA is rapidly generated in one step directly from PCR-generated DNA fragments by cell-free protein expression and in situ immobilisation at a surface. The template for expression is DNA encoding individual proteins or domains, which is produced by PCR using primers designed from information in DNA databases. Coupled transcription and translation is carried out on a surface ...

  6. Quantitation of Cell-Free and Cell-Associated Kaposi's Sarcoma-Associated Herpesvirus DNA by Real-Time PCR

    OpenAIRE

    White, Irene E.; Campbell, Thomas B.

    2000-01-01

    A real-time PCR assay for quantitation of Kaposi's sarcoma-associated herpes virus (KSHV or human herpesvirus 8) DNA was evaluated. The linear dynamic range was 10 to 105 copies of KSHV DNA (r2 > 0.99). The accuracy of DNA purification and quantitation was less than ±0.4 log10 copies for samples that contained from 10 to 105 copies of KSHV DNA. Cell-associated KSHV DNA was quantitated over a range of infected cell frequencies from 0.1 to 10−5, and cell-free KSHV DNA in plasma was quantitated ...

  7. Genome-scale detection of hypermethylated CpG islands in circulating cell-free DNA of hepatocellular carcinoma patients

    OpenAIRE

    Wen, Lu; Li, Jingyi; Guo, Huahu; Liu, Xiaomeng; Zheng, Shengmin; Zhang, Dafang; Zhu, Weihua; Qu, Jianhui; Guo, Limin; Du, Dexiao; Jin, Xiao; Zhang, Yuhao; Gao, Yun; Jie SHEN; Ge, Hao

    2015-01-01

    Despite advances in DNA methylome analyses of cells and tissues, current techniques for genome-scale profiling of DNA methylation in circulating cell-free DNA (ccfDNA) remain limited. Here we describe a methylated CpG tandems amplification and sequencing (MCTA-Seq) method that can detect thousands of hypermethylated CpG islands simultaneously in ccfDNA. This highly sensitive technique can work with genomic DNA as little as 7.5 pg, which is equivalent to 2.5 copies of the haploid genome. We ha...

  8. Graft-Derived Cell-Free DNA as a Marker of Transplant Graft Injury.

    Science.gov (United States)

    Oellerich, Michael; Walson, Philip D; Beck, Julia; Schmitz, Jessica; Kollmar, Otto; Schütz, Ekkehard

    2016-04-01

    Although short-term success after solid organ transplantation is good, long-term graft and recipient survival are both not satisfactory. Despite therapeutic drug monitoring (TDM) of immunosuppressive drugs (ISDs), both excessive and insufficient immunosuppression still do occur. There is a need for new biomarkers that, when combined with TDM, can be used to provide more effective and less toxic, personalized immunosuppression to improve long-term survival. Currently used methods are insufficient to rapidly, cost-effectively, and directly interrogate graft integrity after solid organ transplantation. However, because organ transplants are also genome transplants, measurement of graft-derived circulating cell-free DNA (GcfDNA) has shown promise as a way to improve both graft and recipient outcomes after solid organ transplantation through the early detection of severe graft injury, enabling an early intervention. A newly developed droplet digital polymerase chain reaction (ddPCR) method has advantages over expensive high-throughput sequencing methods to rapidly quantify GcfDNA percentages and absolute amounts. This procedure does not require donor DNA and therefore can be applied to any organ donor/recipient pair. The droplet digital polymerase chain reaction method allows for the early, sensitive, specific, and cost-effective direct assessment of graft integrity and can be used to define individual responses to ISDs including the minimal ISD exposures necessary to prevent rejection. This is especially important in patients undergoing ISD switches due to ISD toxicity, infections, or malignancies. Although prospective, multicenter clinical trials in liver, heart, and kidney transplantation have not been completed, early results suggest that GcfDNA can be combined with TDM to guide changes in immunosuppression to provide more effective, and less toxic treatment. Personalized immunosuppression will shift emphasis in transplantation from reaction to prevention and could

  9. Genome aberrations in canine mammary carcinomas and their detection in cell-free plasma DNA.

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    Julia Beck

    Full Text Available Mammary tumors are the most frequent cancers in female dogs exhibiting a variety of histopathological differences. There is lack of knowledge about the genomes of these common dog tumors. Five tumors of three different histological subtypes were evaluated. Massive parallel sequencing (MPS was performed in comparison to the respective somatic genome of each animal. Copy number and structural aberrations were validated using droplet digital PCR (ddPCR. Using mate-pair sequencing chromosomal aneuploidies were found in two tumors, frequent smaller deletions were found in one, inter-chromosomal fusions in one other, whereas one tumor was almost normal. These aberrations affect several known cancer associated genes such as cMYC, and KIT. One common deletion of the proximal end of CFA27, harboring the tumor suppressor gene PFDN5 was detected in four tumors. Using ddPCR, this deletion was validated and detected in 50% of tumors (N = 20. Breakpoint specific dPCRs were established for four tumors and tumor specific cell-free DNA (cfDNA was detected in the plasma. In one animal tumor-specific cfDNA was found >1 year after surgery, attributable to a lung metastasis. Paired-end sequencing proved that copy-number imbalances of the tumor are reflected by the cfDNA. This report on chromosomal instability of canine mammary cancers reveals similarities to human breast cancers as well as special canine alterations. This animal model provides a framework for using MPS for screening for individual cancer biomarkers with cost effective confirmation and monitoring using ddPCR. The possibility exists that ddPCR can be expanded to screening for common cancer related variants.

  10. Effect of blood pressure and glycemic control on the plasma cell-free DNA in hemodialysis patients

    OpenAIRE

    Jeong, Da Wun; Moon, Ju-Young; Choi, Young-Wook; Moon, Haena; Kim, Kipyo; Lee, Yu-Ho; Kim, Se-Yeun; Kim, Yang-Gyun; Jeong, Kyung-Hwan; Lee, Sang-Ho

    2015-01-01

    Background The plasma levels of cell-free DNA (cfDNA) are known to be elevated under inflammatory or apoptotic conditions. Increased cfDNA levels have been reported in hemodialysis (HD) patients. The aim of this study was to investigate the clinical significance of cfDNA in HD patients. Methods A total of 95 patients on HD were enrolled. We measured their predialysis cfDNA levels using real-time EIF2C1 gene sequence amplification and analyzed its association with certain clinical parameters. ...

  11. Accuracy of non-invasive prenatal testing using cell-free DNA for detection of Down, Edwards and Patau syndromes: a systematic review and meta-analysis

    Science.gov (United States)

    Taylor-Phillips, Sian; Freeman, Karoline; Geppert, Julia; Agbebiyi, Adeola; Uthman, Olalekan A; Madan, Jason; Clarke, Angus; Quenby, Siobhan; Clarke, Aileen

    2016-01-01

    Objective To measure test accuracy of non-invasive prenatal testing (NIPT) for Down, Edwards and Patau syndromes using cell-free fetal DNA and identify factors affecting accuracy. Design Systematic review and meta-analysis of published studies. Data sources PubMed, Ovid Medline, Ovid Embase and the Cochrane Library published from 1997 to 9 February 2015, followed by weekly autoalerts until 1 April 2015. Eligibility criteria for selecting studies English language journal articles describing case–control studies with ≥15 trisomy cases or cohort studies with ≥50 pregnant women who had been given NIPT and a reference standard. Results 41, 37 and 30 studies of 2012 publications retrieved were included in the review for Down, Edwards and Patau syndromes. Quality appraisal identified high risk of bias in included studies, funnel plots showed evidence of publication bias. Pooled sensitivity was 99.3% (95% CI 98.9% to 99.6%) for Down, 97.4% (95.8% to 98.4%) for Edwards, and 97.4% (86.1% to 99.6%) for Patau syndrome. The pooled specificity was 99.9% (99.9% to 100%) for all three trisomies. In 100 000 pregnancies in the general obstetric population we would expect 417, 89 and 40 cases of Downs, Edwards and Patau syndromes to be detected by NIPT, with 94, 154 and 42 false positive results. Sensitivity was lower in twin than singleton pregnancies, reduced by 9% for Down, 28% for Edwards and 22% for Patau syndrome. Pooled sensitivity was also lower in the first trimester of pregnancy, in studies in the general obstetric population, and in cohort studies with consecutive enrolment. Conclusions NIPT using cell-free fetal DNA has very high sensitivity and specificity for Down syndrome, with slightly lower sensitivity for Edwards and Patau syndrome. However, it is not 100% accurate and should not be used as a final diagnosis for positive cases. Trial registration number CRD42014014947. PMID:26781507

  12. Exome Sequencing of Cell-Free DNA from Metastatic Cancer Patients Identifies Clinically Actionable Mutations Distinct from Primary Disease.

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    Timothy M Butler

    Full Text Available The identification of the molecular drivers of cancer by sequencing is the backbone of precision medicine and the basis of personalized therapy; however, biopsies of primary tumors provide only a snapshot of the evolution of the disease and may miss potential therapeutic targets, especially in the metastatic setting. A liquid biopsy, in the form of cell-free DNA (cfDNA sequencing, has the potential to capture the inter- and intra-tumoral heterogeneity present in metastatic disease, and, through serial blood draws, track the evolution of the tumor genome. In order to determine the clinical utility of cfDNA sequencing we performed whole-exome sequencing on cfDNA and tumor DNA from two patients with metastatic disease; only minor modifications to our sequencing and analysis pipelines were required for sequencing and mutation calling of cfDNA. The first patient had metastatic sarcoma and 47 of 48 mutations present in the primary tumor were also found in the cell-free DNA. The second patient had metastatic breast cancer and sequencing identified an ESR1 mutation in the cfDNA and metastatic site, but not in the primary tumor. This likely explains tumor progression on Anastrozole. Significant heterogeneity between the primary and metastatic tumors, with cfDNA reflecting the metastases, suggested separation from the primary lesion early in tumor evolution. This is best illustrated by an activating PIK3CA mutation (H1047R which was clonal in the primary tumor, but completely absent from either the metastasis or cfDNA. Here we show that cfDNA sequencing supplies clinically actionable information with minimal risks compared to metastatic biopsies. This study demonstrates the utility of whole-exome sequencing of cell-free DNA from patients with metastatic disease. cfDNA sequencing identified an ESR1 mutation, potentially explaining a patient's resistance to aromatase inhibition, and gave insight into how metastatic lesions differ from the primary tumor.

  13. Exome Sequencing of Cell-Free DNA from Metastatic Cancer Patients Identifies Clinically Actionable Mutations Distinct from Primary Disease

    Science.gov (United States)

    Butler, Timothy M.; Johnson-Camacho, Katherine; Peto, Myron; Wang, Nicholas J.; Macey, Tara A.; Korkola, James E.; Koppie, Theresa M.; Corless, Christopher L.; Gray, Joe W.; Spellman, Paul T.

    2015-01-01

    The identification of the molecular drivers of cancer by sequencing is the backbone of precision medicine and the basis of personalized therapy; however, biopsies of primary tumors provide only a snapshot of the evolution of the disease and may miss potential therapeutic targets, especially in the metastatic setting. A liquid biopsy, in the form of cell-free DNA (cfDNA) sequencing, has the potential to capture the inter- and intra-tumoral heterogeneity present in metastatic disease, and, through serial blood draws, track the evolution of the tumor genome. In order to determine the clinical utility of cfDNA sequencing we performed whole-exome sequencing on cfDNA and tumor DNA from two patients with metastatic disease; only minor modifications to our sequencing and analysis pipelines were required for sequencing and mutation calling of cfDNA. The first patient had metastatic sarcoma and 47 of 48 mutations present in the primary tumor were also found in the cell-free DNA. The second patient had metastatic breast cancer and sequencing identified an ESR1 mutation in the cfDNA and metastatic site, but not in the primary tumor. This likely explains tumor progression on Anastrozole. Significant heterogeneity between the primary and metastatic tumors, with cfDNA reflecting the metastases, suggested separation from the primary lesion early in tumor evolution. This is best illustrated by an activating PIK3CA mutation (H1047R) which was clonal in the primary tumor, but completely absent from either the metastasis or cfDNA. Here we show that cfDNA sequencing supplies clinically actionable information with minimal risks compared to metastatic biopsies. This study demonstrates the utility of whole-exome sequencing of cell-free DNA from patients with metastatic disease. cfDNA sequencing identified an ESR1 mutation, potentially explaining a patient’s resistance to aromatase inhibition, and gave insight into how metastatic lesions differ from the primary tumor. PMID:26317216

  14. Exome Sequencing of Cell-Free DNA from Metastatic Cancer Patients Identifies Clinically Actionable Mutations Distinct from Primary Disease

    OpenAIRE

    Butler, Timothy M.; Johnson-Camacho, Katherine; Peto, Myron; Wang, Nicholas J.; Macey, Tara A.; Korkola, James E.; Koppie, Theresa M.; Corless, Christopher L.; Joe W. Gray; Spellman, Paul T

    2015-01-01

    The identification of the molecular drivers of cancer by sequencing is the backbone of precision medicine and the basis of personalized therapy; however, biopsies of primary tumors provide only a snapshot of the evolution of the disease and may miss potential therapeutic targets, especially in the metastatic setting. A liquid biopsy, in the form of cell-free DNA (cfDNA) sequencing, has the potential to capture the inter- and intra-tumoral heterogeneity present in metastatic disease, and, thro...

  15. The Impact of Chronic Kidney Disease and Short-Term Treatment with Rosiglitazone on Plasma Cell-Free DNA Levels

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    Amanda L. McGuire

    2014-01-01

    Full Text Available Patients with chronic kidney disease (CKD are at increased risk of cardiovascular disease. Circulating free nucleic acids, known as cell-free DNA (cfDNA, have been proposed as a novel biomarker of cardiovascular risk. The impact of renal impairment on cfDNA levels and whether cfDNA is associated with endothelial dysfunction and inflammation in CKD has not been systematically studied. We analysed cfDNA concentrations from patients with varying degrees of CKD. In addition, to determine whether there is a relationship between cfDNA, inflammation, and endothelial dysfunction in CKD, levels of proinflammatory cytokines and von Willebrand Factor (vWF were measured in patients treated with the peroxisome proliferator-activated receptor gamma agonist rosiglitazone or placebo for 8 weeks. cfDNA levels were not increased with renal impairment or associated with the degree of renal dysfunction (P=0.5. Treatment with rosiglitazone for 8 weeks, but not placebo, was more likely to lead to a reduction in cfDNA levels (P=0.046; however, the absolute changes in cfDNA concentrations during treatment were not statistically significant (P>0.05. cfDNA levels correlated with markers of endothelial dysfunction (hsCRP P=0.0497 and vWF (P=0.0005. In conclusion, cell-free DNA levels are not influenced by renal impairment but do reflect endothelial dysfunction in patients with CKD.

  16. Cell-free DNA in Human Follicular Microenvironment: New Prognostic Biomarker to Predict in vitro Fertilization Outcomes

    OpenAIRE

    Traver, Sabine; Scalici, Elodie; Mullet, Tiffany; Molinari, Nicolas; Vincens, Claire; Anahory, Tal; Hamamah, Samir

    2015-01-01

    Cell-free DNA (cfDNA) fragments, detected in blood and in other biological fluids, are released from apoptotic and/or necrotic cells. CfDNA is currently used as biomarker for the detection of many diseases such as some cancers and gynecological and obstetrics disorders. In this study, we investigated if cfDNA levels in follicular fluid (FF) samples from in vitro fertilization (IVF) patients, could be related to their ovarian reserve status, controlled ovarian stimulation (COS) protocols and I...

  17. Cell-free circulating mitochondrial DNA content and risk of hepatocellular carcinoma in patients with chronic HBV infection

    OpenAIRE

    Ling Li; Hie-Won Hann; Shaogui Wan; Hann, Richard S.; Chun Wang; Yinzhi Lai; Xishan Ye; Alison Evans; Ronald E Myers; Zhong Ye; Bingshan Li; Jinliang Xing; Hushan Yang

    2016-01-01

    Recent studies have demonstrated a potential link between circulating cell-free mitochondrial DNA (mtDNA) content and cancers. However, there is no study evaluating the association between circulating mtDNA as a non-invasive marker of hepatocellular carcinoma (HCC) risk. We conducted a nested case-control study to determine circulating mtDNA content in serum samples from 116 HBV-related HCC cases and 232 frequency-matched cancer-free HBV controls, and evaluate the retrospective association be...

  18. Urine Cell-Free DNA Integrity as a Marker for Early Prostate Cancer Diagnosis: A Pilot Study

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    Valentina Casadio

    2013-01-01

    Full Text Available Circulating cell-free DNA has been recognized as an accurate marker for the diagnosis of prostate cancer, whereas the role of urine cell-free DNA (UCF DNA has never been evaluated in this setting. It is known that normal apoptotic cells produce highly fragmented DNA while cancer cells release longer DNA. We thus verified the potential role of UCF DNA integrity for early prostate cancer diagnosis. UCF DNA was isolated from 29 prostate cancer patients and 25 healthy volunteers. Sequences longer than 250 bp (c-Myc, BCAS1, and HER2 were quantified by real-time PCR to verify UCF DNA integrity. Receiver operating characteristic (ROC curve analysis revealed an area under the curve of 0.7959 (95% CI 0.6729–0.9188. At the best cut-off value of 0.04 ng/μL, UCF DNA integrity analysis showed a sensitivity of 0.79 (95% CI 0.62–0.90 and a specificity of 0.84 (95% CI 0.65–0.94. These preliminary findings indicate that UCF DNA integrity could be a promising noninvasive marker for the early diagnosis of prostate cancer and pave the way for further research into this area.

  19. Diagnosing schistosomiasis by detection of cell-free parasite DNA in human plasma.

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    Dominic Wichmann

    Full Text Available INTRODUCTION: Schistosomiasis (bilharzia, one of the most relevant parasitoses of humans, is confirmed by microscopic detection of eggs in stool, urine, or organ biopsies. The sensitivity of these procedures is variable due to fluctuation of egg shedding. Serological tests on the other hand do not distinguish between active and past disease. In patients with acute disease (Katayama syndrome, both serology and direct detection may produce false negative results. To overcome these obstacles, we developed a novel diagnostic strategy, following the rationale that Schistosoma DNA may be liberated as a result of parasite turnover and reach the blood. Cell-free parasite DNA (CFPD was detected in plasma by PCR. METHODOLOGY/PRINCIPAL FINDINGS: Real-time PCR with internal control was developed and optimized for detection of CFPD in human plasma. Distribution was studied in a mouse model for Schistosoma replication and elimination, as well as in human patients seen before and after treatment. CFPD was detectable in mouse plasma, and its concentration correlated with the course of anti-Schistosoma treatment. Humans with chronic disease and eggs in stool or urine (n = 14 showed a 100% rate of CFPD detection. CFPD was also detected in all (n = 8 patients with Katayama syndrome. Patients in whom no viable eggs could be detected and who had been treated for schistomiasis in the past (n = 30 showed lower detection rates (33.3% and significantly lower CFPD concentrations. The duration from treatment to total elimination of CFPD from plasma was projected to exceed one year. CONCLUSIONS/SIGNIFICANCE: PCR for detection of CFPD in human plasma may provide a new laboratory tool for diagnosing schistosomiasis in all phases of clinical disease, including the capacity to rule out Katayama syndrome and active disease. Further studies are needed to confirm the clinical usefulness of CFPD quantification in therapy monitoring.

  20. Genome-wide DNA Methylation Profiling of Cell-Free Serum DNA in Esophageal Adenocarcinoma and Barrett Esophagus

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    Rihong Zhai

    2012-01-01

    Full Text Available Aberrant DNA methylation (DNAm is a feature of most types of cancers. Genome-wide DNAm profiling has been performed successfully on tumor tissue DNA samples. However, the invasive procedure limits the utility of tumor tissue for epidemiological studies. While recent data indicate that cell-free circulating DNAm (cfDNAm profiles reflect DNAm status in corresponding tumor tissues, no studies have examined the association of cfDNAm with cancer or precursors on a genome-wide scale. The objective of this pilot study was to evaluate the putative significance of genome-wide cfDNAm profiles in esophageal adenocarcinoma (EA and Barrett esophagus (BE, EA precursor. We performed genome-wide DNAm profiling in EA tissue DNA (n = 8 and matched serum DNA (n = 8, in serum DNA of BE (n = 10, and in healthy controls (n = 10 using the Infinium HumanMethylation27 BeadChip that covers 27,578 CpG loci in 14,495 genes. We found that cfDNAm profiles were highly correlated to DNAm profiles in matched tumor tissue DNA (r = 0.92 in patients with EA. We selected the most differentially methylated loci to perform hierarchical clustering analysis. We found that 911 loci can discriminate perfectly between EA and control samples, 554 loci can separate EA from BE samples, and 46 loci can distinguish BE from control samples. These results suggest that genome-wide cfDNAm profiles are highly consistent with DNAm profiles detected in corresponding tumor tissues. Differential cfDNAm profiling may be a useful approach for the noninvasive screening of EA and EA premalignant lesions.

  1. Cell-free mitochondrial DNA in CSF is associated with early viral rebound, inflammation, and severity of neurocognitive deficits in HIV infection

    OpenAIRE

    Pérez-Santiago, J; Schrier, RD; de Oliveira, MF; Gianella, S; Var, SR; Day, TRC; Ramirez-Gaona, M; Suben, JD; Murrell, B.; Massanella, M; Cherner, M.; Smith, DM; Ellis, RJ; Letendre, SL; Mehta, SR

    2016-01-01

    © 2015 Journal of NeuroVirology, Inc. Cell-free mitochondiral DNA (mtDNA) is an immunogenic molecule associated with many inflammatory conditions. We evaluated the relationship between cell-free mtDNA in cerebrospinal fluid (CSF) and neurocognitive performance and inflammation during HIV infection. In a cross-sectional analysis, we evaluated the association of mtDNA levels with clinical assessments, inflammatory markers, and neurocognitive performance in 28 HIV-infected individuals. In CSF, w...

  2. A cell-free system for studying a priming factor involved in repair of bleomycin-damaged DNA.

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    Seki,Shuji

    1989-04-01

    Full Text Available A simple cell-free system for studying a priming factor involved in the repair of bleomycin-damaged DNA was established. The template-primer used for the repair DNA synthesis was prepared by treating the closed circular, superhelical form of pUC19 plasmid DNA with 2.2 microM bleomycin and 20 microM ferrous ions. Single-strand breaks were introduced into pUC19 DNA by the bleomycin treatment, and the DNA was consequently converted largely into the open circular form. A system for repair of this bleomycin-damaged DNA was constructed with a priming factor, DNA polymerase (DNA polymerase beta or Klenow fragment of DNA polymerase I, ATP, T4 DNA ligase and four deoxynucleoside triphosphates. After incubation, the conformation of the DNA was analyzed by agarose gel electrophoresis and electron microscopy. The open circular DNA was largely converted to the closed circular DNA, indicating that the single-strand breaks of DNA were repaired. When the priming factor was omitted, DNA repair did not occur. The present system seemed to be applicable to the study of priming factors involved in the repair of DNA with single-strand breaks caused not only by bleomycin but also by ionizing radiation or active oxygen.

  3. A1M Ameliorates Preeclampsia-Like Symptoms in Placenta and Kidney Induced by Cell-Free Fetal Hemoglobin in Rabbit.

    Science.gov (United States)

    Nääv, Åsa; Erlandsson, Lena; Axelsson, Josefin; Larsson, Irene; Johansson, Martin; Wester-Rosenlöf, Lena; Mörgelin, Matthias; Casslén, Vera; Gram, Magnus; Åkerström, Bo; Hansson, Stefan R

    2015-01-01

    Preeclampsia is one of the most serious pregnancy-related diseases and clinically manifests as hypertension and proteinuria after 20 gestational weeks. The worldwide prevalence is 3-8% of pregnancies, making it the most common cause of maternal and fetal morbidity and mortality. Preeclampsia lacks an effective therapy, and the only "cure" is delivery. We have previously shown that increased synthesis and accumulation of cell-free fetal hemoglobin (HbF) in the placenta is important in the pathophysiology of preeclampsia. Extracellular hemoglobin (Hb) and its metabolites induce oxidative stress, which may lead to acute renal failure and vascular dysfunction seen in preeclampsia. The human endogenous protein, α1-microglobulin (A1M), removes cell-free heme-groups and induces natural tissue repair mechanisms. Exogenously administered A1M has been shown to alleviate the effects of Hb-induced oxidative stress in rat kidneys. Here we attempted to establish an animal model mimicking the human symptoms at stage two of preeclampsia by administering species-specific cell-free HbF starting mid-gestation until term, and evaluated the therapeutic effect of A1M on the induced symptoms. Female pregnant rabbits received HbF infusions i.v. with or without A1M every second day from gestational day 20. The HbF-infused animals developed proteinuria and a significantly increased glomerular sieving coefficient in kidney that was ameliorated by co-administration of A1M. Transmission electron microscopy analysis of kidney and placenta showed both intracellular and extracellular tissue damages after HbF-treatment, while A1M co-administration resulted in a significant reduction of the structural and cellular changes. Neither of the HbF-treated animals displayed any changes in blood pressure during pregnancy. In conclusion, infusion of cell-free HbF in the pregnant rabbits induced tissue damage and organ failure similar to those seen in preeclampsia, and was restored by co-administration of A

  4. A1M Ameliorates Preeclampsia-Like Symptoms in Placenta and Kidney Induced by Cell-Free Fetal Hemoglobin in Rabbit.

    Directory of Open Access Journals (Sweden)

    Åsa Nääv

    Full Text Available Preeclampsia is one of the most serious pregnancy-related diseases and clinically manifests as hypertension and proteinuria after 20 gestational weeks. The worldwide prevalence is 3-8% of pregnancies, making it the most common cause of maternal and fetal morbidity and mortality. Preeclampsia lacks an effective therapy, and the only "cure" is delivery. We have previously shown that increased synthesis and accumulation of cell-free fetal hemoglobin (HbF in the placenta is important in the pathophysiology of preeclampsia. Extracellular hemoglobin (Hb and its metabolites induce oxidative stress, which may lead to acute renal failure and vascular dysfunction seen in preeclampsia. The human endogenous protein, α1-microglobulin (A1M, removes cell-free heme-groups and induces natural tissue repair mechanisms. Exogenously administered A1M has been shown to alleviate the effects of Hb-induced oxidative stress in rat kidneys. Here we attempted to establish an animal model mimicking the human symptoms at stage two of preeclampsia by administering species-specific cell-free HbF starting mid-gestation until term, and evaluated the therapeutic effect of A1M on the induced symptoms. Female pregnant rabbits received HbF infusions i.v. with or without A1M every second day from gestational day 20. The HbF-infused animals developed proteinuria and a significantly increased glomerular sieving coefficient in kidney that was ameliorated by co-administration of A1M. Transmission electron microscopy analysis of kidney and placenta showed both intracellular and extracellular tissue damages after HbF-treatment, while A1M co-administration resulted in a significant reduction of the structural and cellular changes. Neither of the HbF-treated animals displayed any changes in blood pressure during pregnancy. In conclusion, infusion of cell-free HbF in the pregnant rabbits induced tissue damage and organ failure similar to those seen in preeclampsia, and was restored by co

  5. Cell-free DNA concentration and integrity as a screening tool for cancer

    Directory of Open Access Journals (Sweden)

    Ebtsam R Zaher

    2013-01-01

    Full Text Available Aim of the Study: This study aims to evaluate cell-free DNA (CFDNA concentration and integrity in patients with malignant and nonmalignant diseases and in controls to investigate their value as a screening test for cancer, and to correlate them with clinicopathological parameters of cancer patients. Materials and Methods: The study included three groups; group I: 120 cancer patients, group II: 120 patients with benign diseases and group III: 120 normal healthy volunteers as control. One plasma sample was collected from each subject. CFDNA was purified from the plasma then its concentration was measured and integrity was assessed by PCR amplification of 100, 200, 400, and 800 bp bands. Results: There was a highly significant difference in CFDNA levels between cancer group and each of benign and control groups. AUC of ROC curve for cancer group versus normal and benign groups were 0.962 and 0.895, which indicated the efficiency of CFDNA as a marker of cancer. As for integrity, normal and benign subjects showed only two bands at 100 and 200 bp, while all cancer patients demonstrated the 400 bp band and 78% of them had the 800 bp whose presence correlated with vascular invasion. Conclusion: The combined use of CFDNA concentration and integrity is a candidate for a universal screening test of cancer. Upon setting suitable boundaries for the test it might be applied to identify cancer patients, particularly among subjects with predisposing factors. Being less expensive, CFDNA concentration could be applied for mass screening and for patients with values overlapping those of normal and benign subjects, the use of the more expensive, yet more specific, integrity test is suggested.

  6. Smoking During Pregnancy Seems to Alter Fetal DNA, Study Finds

    Science.gov (United States)

    ... nlm.nih.gov/medlineplus/news/fullstory_158077.html Smoking During Pregnancy Seems to Alter Fetal DNA, Study ... that were affected by a mother-to-be's smoking. The findings may help improve understanding about the ...

  7. Actionable mutations in plasma cell-free DNA in patients with advanced cancers referred for experimental targeted therapies

    OpenAIRE

    Janku, Filip; Angenendt, Philipp; Tsimberidou, Apostolia M.; Fu, Siqing; Naing, Aung; Falchook, Gerald S.; David S Hong; Holley, Veronica R.; Cabrilo, Goran; Jennifer J Wheler; Piha-Paul, Sarina A.; Zinner, Ralph G.; Bedikian, Agop Y.; Overman, Michael J.; Kee, Bryan K.

    2015-01-01

    Cell-free (cf) DNA in the plasma of cancer patients offers an easily obtainable source of biologic material for mutation analysis. Plasma samples from 157 patients with advanced cancers who progressed on systemic therapy were tested for 21 mutations in BRAF, EGFR, KRAS, and PIK3CA using the BEAMing method and results were compared to mutation analysis of archival tumor tissue from a CLIA-certified laboratory obtained as standard of care from diagnostic or therapeutic procedures. Results were ...

  8. Increased Plasma Cell-Free DNA Level during HTNV Infection: Correlation with Disease Severity and Virus Load

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    Jing Yi

    2014-07-01

    Full Text Available Cell-free DNA (cf-DNA in blood represents a promising DNA damage response triggered by virus infection or trauma, tumor, etc. Hantavirus primarily causes two diseases: haemorrhagic fever with renal syndrome (HFRS and Hantavirus cardiopulmonary syndrome (HCPS, depending on different Hantavirus species. The aim of this study was to evaluate plasma cf-DNA levels in acute phase of HFRS, and to correlate plasma cf-DNA with disease severity and plasma Hanttan virus (HTNV load. We observed the appearance of cf-DNA in 166 plasma samples from 76 HFRS patients: the plasma cf-DNA levels peaked at the hypotensive stage of HFRS, and then decreased gradually. Until the diuretic stage, there was no significant difference in plasma cf-DNA level between patients and the healthy control. Exclusively in the febrile/hypotensive stage, the plasma cf-DNA levels of severe/critical patients were higher than those of the mild/moderate group. Moreover, the plasma cf-DNA value in the early stage of HFRS was correlated with HTNV load and disease severity. In most of the patients, plasma cf-DNA displayed a low-molecular weight appearance, corresponding to the size of apoptotic DNA. In conclusion, the plasma cf-DNA levels were dynamically elevated during HFRS, and correlated with disease severity, which suggests that plasma cf-DNA may be a potential biomarker for the pathogenesis and prognosis of HFRS.

  9. KRAS G12V Mutation Detection by Droplet Digital PCR in Circulating Cell-Free DNA of Colorectal Cancer Patients

    OpenAIRE

    Olmedillas López, Susana; García-Olmo, Dolores C; García-Arranz, Mariano; Guadalajara, Héctor; Pastor, Carlos; García-Olmo, Damián

    2016-01-01

    KRAS mutations are responsible for resistance to anti-epidermal growth factor receptor (EGFR) therapy in colorectal cancer patients. These mutations sometimes appear once treatment has started. Detection of KRAS mutations in circulating cell-free DNA in plasma (“liquid biopsy”) by droplet digital PCR (ddPCR) has emerged as a very sensitive and promising alternative to serial biopsies for disease monitoring. In this study, KRAS G12V mutation was analyzed by ddPCR in plasma DNA from 10 colorect...

  10. Effect of blood pressure and glycemic control on the plasma cell-free DNA in hemodialysis patients

    Science.gov (United States)

    Jeong, Da Wun; Moon, Ju-Young; Choi, Young-Wook; Moon, Haena; Kim, Kipyo; Lee, Yu-Ho; Kim, Se-Yeun; Kim, Yang-Gyun; Jeong, Kyung-Hwan; Lee, Sang-Ho

    2015-01-01

    Background The plasma levels of cell-free DNA (cfDNA) are known to be elevated under inflammatory or apoptotic conditions. Increased cfDNA levels have been reported in hemodialysis (HD) patients. The aim of this study was to investigate the clinical significance of cfDNA in HD patients. Methods A total of 95 patients on HD were enrolled. We measured their predialysis cfDNA levels using real-time EIF2C1 gene sequence amplification and analyzed its association with certain clinical parameters. Results The mean plasma cfDNA level in the HD patients was 3,884 ± 407 GE/mL, and the mean plasma cfDNA level in the control group was 1,420 ± 121 GE/mL (P < 0.05). Diabetic patients showed higher plasma cfDNA levels compared with nondiabetic patients (P < 0.01). Patients with cardiovascular complications also showed higher plasma cfDNA levels compared with those without cardiovascular complication (P < 0.05). In univariable analysis, the cfDNA level was associated with 3-month mean systolic blood pressure (SBP), white blood cell, serum albumin, creatinine (Cr), normalized protein catabolic rate in HD patients. In diabetic patients, it was significantly correlated with SBP, hemoglobin A1c, and serum albumin. In multivariate analysis, SBP was the independent determinant for the cfDNA level. In diabetic patients, cfDNA level was independently associated with hemoglobin A1c and SBP. Conclusions In patients with HD, cfDNA is elevated in diabetic patients and patients with cardiovascular diseases. Uncontrolled hypertension and poor glycemic control are independent determinants for the elevated cfDNA. Our data suggest that cfDNA might be a marker of vascular injury rather than proinflammatory condition in HD patients. PMID:26779422

  11. Cell-free fetal DNA detection in maternal plasma using real-time PCR and cycling probe technology for prenatal screening β-thalassaemia major%实时PCR和cycling probe技术检测母血浆游离胎儿DNA筛选重型β地中海贫血胎儿

    Institute of Scientific and Technical Information of China (English)

    陈熙; 任景慧; 郭辉; 林琳华; 姚秋璇

    2008-01-01

    目的 通过检测孕妇外周血中的游离胎儿DNA来筛选重型β-地中海贫血胎儿.方法 选择行产前基因诊断的夫妇6对,孕妇孕周23~26周.血液学检查:胎儿的父亲均为β-地中海贫血17M/N型,孕妇本人为携带除17M/N型之外的另一β-地中海贫血突变类型.针对CD17(A→T)无义突变,设计β-珠蛋白肽链上该等位基因的一对特异性引物和通过cycling probe法分别设计检测正常基因序列和基因突变位点的两条荧光探针,分别用FAM和HEX荧光标记.结合RT-PCR技术检测孕妇外周血中游离胎儿DNA,诊断胎儿是否遗传了其父亲的β地中海贫血17M/N碱基突变位点.同时与脐血血液学检查所诊断的胎儿地贫基因型对照.结果 提取的6例孕妇血浆DNA模板中有3例同时显示FAM和HEX荧光信号值阳性结果,即这3例孕妇的胎儿遗传了父亲β-珠蛋白肽链上CD17位点的突变碱基(A→T).另外3例孕妇血浆DNA模板的FAM信号值阳性,HEX信号值阴性,即所孕胎儿没有遗传父亲的CD17位点的突变碱基.结论 利用RT-PCR和cycling probe技术检测孕妇外周血中的游离胎儿DNA可用来筛选患重型地中海贫血的胎儿.

  12. Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

    Directory of Open Access Journals (Sweden)

    Peng Xia

    2009-01-01

    Full Text Available Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf mitochondrial (mtDNA and nuclear (nDNA DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency.

  13. Relationship of plasma cell-free DNA level with mortality and prognosis in patients with Crimean-Congo hemorrhagic fever.

    Science.gov (United States)

    Bakir, Mehmet; Engin, Aynur; Kuskucu, Mert Ahmet; Bakir, Sevtap; Gündag, Omür; Midilli, Kenan

    2016-07-01

    Crimean-Congo hemorrhagic fever (CCHF) is a viral infection. Circulating plasma cell-free DNA (pcf-DNA) is a novel marker indicating cellular damage. So far, the role of pcf-DNA did not investigate in CCHF patients. In the current study, pcf-DNA levels were investigated in CCHF patients with different clinical severity grades to explore the relationship between circulating pcf-DNA level, virus load, and disease severity. Seventy-two patients were categorized as mild, intermediate, and severe based on severity grading scores. The pcf-DNA level was obtained from all participants on admission and from the survivors on the day of the discharge. The controls consisted of 31 healthy. Although the pcf-DNA level at admission was higher in patients than in the controls, the difference was not statistically significant (P = 0.291). However, at admission and in the convalescent period, the difference between pcf-DNA levels in mild, intermediate, and severe patient groups was significant. The pcf-DNA level in severe patients was higher than in the others. Furthermore, compared to survivors, non-survivors had higher pcf-DNA levels at admission (P = 0.001). A direct relationship was found between the pcf-DNA level and the viral load on the day of discharge in surviving patients. ROC curve analysis identified a pcf-DNA level of 0.42 as the optimal cut-off for prediction of mortality. The positive predictive value, negative predictive value, specificity, and sensitivity for predicting mortality was 100%, 72%, 100%, and 79%, respectively. In summary, our findings revealed that pcf-DNA levels may be used as a biomarker in predicting CHHF prognosis. J. Med. Virol. 88:1152-1158, 2016. © 2015 Wiley Periodicals, Inc. PMID:26680021

  14. Quantitative cell-free DNA, KRAS, and BRAF mutations in plasma from patients with metastatic colorectal cancer during treatment with cetuximab and irinotecan

    DEFF Research Database (Denmark)

    Spindler, Karen-Lise Garm; Pallisgaard, Niels; Vogelius, Ivan Storgaard; Jakobsen, Anders

    2012-01-01

    The present study investigated the levels of circulating cell-free DNA (cfDNA) in plasma from patients with metastatic colorectal cancer (mCRC) in relation to third-line treatment with cetuximab and irinotecan and the quantitative relationship of cfDNA with tumor-specific mutations in plasma....

  15. Branched DNA-based Alu quantitative assay for cell-free plasma DNA levels in patients with sepsis or systemic inflammatory response syndrome.

    Science.gov (United States)

    Hou, Yan-Qiang; Liang, Dong-Yu; Lou, Xiao-Li; Zhang, Mei; Zhang, Zhen-huan; Zhang, Lu-rong

    2016-02-01

    Cell-free circulating DNA (cf-DNA) can be detected by various of laboratory techniques. We described a branched DNA-based Alu assay for measuring cf-DNA in septic patients. Compared to healthy controls and systemic inflammatory response syndrome (SIRS) patients, serum cf-DNA levels were significantly higher in septic patients (1426.54 ± 863.79 vs 692.02 ± 703.06 and 69.66 ± 24.66 ng/mL). The areas under the receiver operating characteristic curve of cf-DNA for normal vs sepsis and SIRS vs sepsis were 0.955 (0.884-1.025), and 0.856 (0.749-0.929), respectively. There was a positive correlation between cf-DNA and interleukin 6 or procalcitonin or Acute Physiology and Chronic Health Evaluation II. The cf-DNA concentration was higher in intensive care unit nonsurviving patients compared to surviving patients (2183.33 ± 615.26 vs 972.46 ± 648.36 ng/mL; P format. Cell-free circulating DNA might be a new marker in discrimination of sepsis and SIRS. PMID:26589770

  16. Aberrant reduction of telomere repetitive sequences in plasma cell-free DNA for early breast cancer detection

    Science.gov (United States)

    Wu, Xi; Tanaka, Hiromi

    2015-01-01

    Excessive telomere shortening is observed in breast cancer lesions when compared to adjacent non-cancerous tissues, suggesting that telomere length may represent a key biomarker for early cancer detection. Because tumor-derived, cell-free DNA (cfDNA) is often released from cancer cells and circulates in the bloodstream, we hypothesized that breast cancer development is associated with changes in the amount of telomeric cfDNA that can be detected in the plasma. To test this hypothesis, we devised a novel, highly sensitive and specific quantitative PCR (qPCR) assay, termed telomeric cfDNA qPCR, to quantify plasma telomeric cfDNA levels. Indeed, the internal reference primers of our design correctly reflected input cfDNA amount (R2 = 0.910, P = 7.82 × 10−52), implying accuracy of this assay. We found that plasma telomeric cfDNA levels decreased with age in healthy individuals (n = 42, R2 = 0.094, P = 0.048), suggesting that cfDNA is likely derived from somatic cells in which telomere length shortens with increasing age. Our results also showed a significant decrease in telomeric cfDNA level from breast cancer patients with no prior treatment (n = 47), compared to control individuals (n = 42) (P = 4.06 × 10−8). The sensitivity and specificity for the telomeric cfDNA qPCR assay was 91.49% and 76.19%, respectively. Furthermore, the telomeric cfDNA level distinguished even the Ductal Carcinoma In Situ (DCIS) group (n = 7) from the healthy group (n = 42) (P = 1.51 × 10−3). Taken together, decreasing plasma telomeric cfDNA levels could be an informative genetic biomarker for early breast cancer detection. PMID:26356673

  17. Cell-free DNA Fragmentation Patterns in Amniotic Fluid Identify Genetic Abnormalities and Changes due to Storage

    Science.gov (United States)

    Peter, Inga; Tighiouart, Hocine; Lapaire, Olav; Johnson, Kirby L.; Bianchi, Diana W.; Terrin, Norma

    2015-01-01

    Circulating cell-free DNA (cfDNA) has become a promising biomarker in prenatal diagnosis. However, despite extensive studies in different body fluids, cfDNA predictive value is uncertain owing to the confounding factors that can affect its levels, such as gestational age, maternal weight, smoking status, and medications. Residual fresh and archived amniotic fluid (AF) supernatants were obtained from gravid women (mean gestational age 17 wk) carrying euploid (N = 36) and aneuploid (N = 29) fetuses, to characterize cfDNA-fragmentation patterns with regard to aneuploidy and storage time (−80°C). AF cfDNA was characterized by the real-time quantitative polymerase chain reaction amplification of glyceraldehyde-3-phosphate dehydrogenase, gel electrophoresis, and pattern recognition of the DNA fragmentation. The distributions of cfDNA fragment lengths were compared using 6 measures that defined the locations and slopes for the first and last peaks, after elimination of the confounding variables. This method allowed for the unique classification of euploid and aneuploid cfDNA samples in AF, which had been matched for storage time. In addition, we showed that archived euploid AF samples gradually lose long cfDNA fragments: this loss accurately distinguishes them from the fresh samples. We present preliminary data using cfDNA-fragmentation patterns, to uniquely distinguish between AF samples of pregnant women with regard to aneuploidy and storage time, independent of gestational age and initial DNA amount. In addition to potential applications in prenatal diagnosis, these data suggest that archived AF samples consist of large amounts of short cfDNA fragments, which are undetectable using standard real-time polymerase chain reaction amplification. PMID:18382362

  18. A New Model for Providing Cell-Free DNA and Risk Assessment for Chromosome Abnormalities in a Public Hospital Setting

    Directory of Open Access Journals (Sweden)

    Robert Wallerstein

    2014-01-01

    Full Text Available Objective. Cell-free DNA (cfDNA offers highly accurate noninvasive screening for Down syndrome. Incorporating it into routine care is complicated. We present our experience implementing a novel program for cfDNA screening, emphasizing patient education, genetic counseling, and resource management. Study Design. Beginning in January 2013, we initiated a new patient care model in which high-risk patients for aneuploidy received genetic counseling at 12 weeks of gestation. Patients were presented with four pathways for aneuploidy risk assessment and diagnosis: (1 cfDNA; (2 integrated screening; (3 direct-to-invasive testing (chorionic villus sampling or amniocentesis; or (4 no first trimester diagnostic testing/screening. Patients underwent follow-up genetic counseling and detailed ultrasound at 18–20 weeks to review first trimester testing and finalize decision for amniocentesis. Results. Counseling and second trimester detailed ultrasound were provided to 163 women. Most selected cfDNA screening (69% over integrated screening (0.6%, direct-to-invasive testing (14.1%, or no screening (16.6%. Amniocentesis rates decreased following implementation of cfDNA screening (19.0% versus 13.0%, P<0.05. Conclusion. When counseled about screening options, women often chose cfDNA over integrated screening. This program is a model for patient-directed, efficient delivery of a newly available high-level technology in a public health setting. Genetic counseling is an integral part of patient education and determination of plan of care.

  19. Cell-free DNA Comprises an In Vivo Nucleosome Footprint that Informs Its Tissues-Of-Origin.

    Science.gov (United States)

    Snyder, Matthew W; Kircher, Martin; Hill, Andrew J; Daza, Riza M; Shendure, Jay

    2016-01-14

    Nucleosome positioning varies between cell types. By deep sequencing cell-free DNA (cfDNA), isolated from circulating blood plasma, we generated maps of genome-wide in vivo nucleosome occupancy and found that short cfDNA fragments harbor footprints of transcription factors. The cfDNA nucleosome occupancies correlate well with the nuclear architecture, gene structure, and expression observed in cells, suggesting that they could inform the cell type of origin. Nucleosome spacing inferred from cfDNA in healthy individuals correlates most strongly with epigenetic features of lymphoid and myeloid cells, consistent with hematopoietic cell death as the normal source of cfDNA. We build on this observation to show how nucleosome footprints can be used to infer cell types contributing to cfDNA in pathological states such as cancer. Since this strategy does not rely on genetic differences to distinguish between contributing tissues, it may enable the noninvasive monitoring of a much broader set of clinical conditions than currently possible. PMID:26771485

  20. Plasma cell-free DNA levels and integrity in patients with chest radiological findings: NSCLC versus benign lung nodules.

    Science.gov (United States)

    Szpechcinski, Adam; Rudzinski, Piotr; Kupis, Wlodzimierz; Langfort, Renata; Orlowski, Tadeusz; Chorostowska-Wynimko, Joanna

    2016-05-01

    Effective discrimination between lung cancer and benign tumours is a common clinical problem in the differential diagnosis of solitary pulmonary nodules. The analysis of cell-free DNA (cfDNA) in blood may greatly aid the early detection of lung cancer by evaluating cancer-related alterations. The plasma cfDNA levels and integrity were analysed in 65 non-small cell lung cancer (NSCLC) patients, 28 subjects with benign lung tumours, and 16 healthy controls using real-time PCR. The NSCLC patients demonstrated significantly higher mean plasma cfDNA levels compared with those with benign tumours (P = 0.0009) and healthy controls (P 2.8 ng/ml provided 86.4% sensitivity and 61.4% specificity in discriminating NSCLC from benign lung pathologies and healthy controls. cfDNA integrity showed better discriminatory power (91% sensitivity, 68.2% specificity). These data demonstrate that plasma cfDNA concentration and integrity analyses can significantly differentiate between NSCLC and benign lung tumours. The diagnostic capacity of the quantitative cfDNA assay is comparable to the values presented by conventional imaging modalities used in clinical practice. PMID:26854716

  1. Cell-free circulating mitochondrial DNA content and risk of hepatocellular carcinoma in patients with chronic HBV infection.

    Science.gov (United States)

    Li, Ling; Hann, Hie-Won; Wan, Shaogui; Hann, Richard S; Wang, Chun; Lai, Yinzhi; Ye, Xishan; Evans, Alison; Myers, Ronald E; Ye, Zhong; Li, Bingshan; Xing, Jinliang; Yang, Hushan

    2016-01-01

    Recent studies have demonstrated a potential link between circulating cell-free mitochondrial DNA (mtDNA) content and cancers. However, there is no study evaluating the association between circulating mtDNA as a non-invasive marker of hepatocellular carcinoma (HCC) risk. We conducted a nested case-control study to determine circulating mtDNA content in serum samples from 116 HBV-related HCC cases and 232 frequency-matched cancer-free HBV controls, and evaluate the retrospective association between mtDNA content and HCC risk using logistic regression and their temporal relationship using a mixed effects model. HCC cases had significantly lower circulating mtDNA content than controls (1.06 versus 2.47, P = 1.7 × 10(-5)). Compared to HBV patients with higher mtDNA content, those with lower mtDNA content had a significantly increased risk of HCC with an odds ratio (OR) of 2.19 (95% confidence interval [CI] 1.28-3.72, P = 0.004). Quartile analyses revealed a significant dose-dependent effect (Ptrend = 0.001) for this association. In a pilot longitudinal sub-cohort of 14 matched cases-control pairs, we observed a trend of dramatically decreased mtDNA content in cases and slightly decreased mtDNA content in controls, with a significant interaction of case-control status with time (Pinteraction = 0.049). Our findings suggest that circulating mtDNA is a potential novel non-invasive biomarker of HCC risk in HBV patients. PMID:27063412

  2. Chimerism Analysis of Cell-Free DNA in Patients Treated with Hematopoietic Stem Cell Transplantation May Predict Early Relapse in Patients with Hematologic Malignancies

    OpenAIRE

    Mahmoud Aljurf; Hala Abalkhail; Amal Alseraihy; Said Y. Mohamed; Mouhab Ayas; Fahad Alsharif; Hazza Alzahrani; Abdullah Al-Jefri; Ghuzayel Aldawsari; Ali Al-Ahmari; Belgaumi, Asim F.; Claudia Ulrike Walter; Hassan El-Solh; Walid Rasheed; Maher Albitar

    2016-01-01

    Background. We studied DNA chimerism in cell-free DNA (cfDNA) in patients treated with HSCT. Methods. Chimerism analysis was performed on CD3+ cells, polymorphonuclear (PMN) cells, and cfDNA using 16 small tandem repeat loci. The resulting labeled PCR-products were size-fractionated and quantified. Results. Analyzing samples from 191 patients treated with HSCT for nonneoplastic hematologic disorders demonstrated that the cfDNA chimerism is comparable to that seen in PMN cells. Analyzing leuke...

  3. Efficient Capture and Isolation of Tumor-Related Circulating Cell-Free DNA from Cancer Patients Using Electroactive Conducting Polymer Nanowire Platforms

    OpenAIRE

    Jeon, SeungHyun; Lee, HyungJae; Bae, Kieun; Yoon, Kyong-Ah; Lee, Eun Sook; Cho, Youngnam

    2016-01-01

    Circulating cell-free DNA (cfDNA) is currently recognized as a key non-invasive biomarker for cancer diagnosis and progression and therapeutic efficacy monitoring. Because cfDNA has been detected in patients with diverse types of cancers, the use of efficient strategies to isolate cfDNA not only provides valuable insights into tumour biology, but also offers the potential for developing new cancer-specific targets. However, the challenges associated with conventional cfDNA extraction methods ...

  4. O6-methylguanine DNA methyltransferase in human fetal tissues: fetal and maternal factors

    International Nuclear Information System (INIS)

    O6-Methylguanine methyltransferase (O6-MT) was measured and compared in extracts of 7 human fetal tissues obtained from 21 different fetal specimens as a function of fetal age and race, and maternal smoking and drug usage. Activity was determined from the proteinase-K solubilized radioactivity transferred from the DNA to the O6-MT. S9 homogenates were incubated with a heat depurinated [3H]-methylnitrosourea alkylated DNA. Liver exhibited the highest activity followed by kidney, lung, small intestine, large intestine, skin and brain. Each of the tissues exhibited a 3- to 5-fold level of interindividual variation of O6-MT. There did not appear to be any significant difference of O6-MT in the tissues obtained from mothers who smoked cigarettes during pregnancy. Also, fetal race and age did not appear to account for the level of variation of O6-MT. The fetal tissues obtained from an individual using phenobarbital and smoking exhibited 4-fold increases in O6-MT activity. The tissues obtained from another individual on kidney dialysis were 2- to 3-fold higher than the normal population. These data suggest that the variation in human O6-MT can not be explained by racial or smoking factors, but may be modulated by certain drugs

  5. Prospective blinded study of somatic mutation detection in cell-free DNA utilizing a targeted 54-gene next generation sequencing panel in metastatic solid tumor patients

    OpenAIRE

    Kim, Seung Tae; Lee, Won Suk; Lanman, Richard B.; Mortimer, Stefanie; Zill, Oliver A.; Kim, Kyoung-Mee; Jang, Kee Taek; Kim, Seok-Hyung; Park, Se Hoon; Park, Joon Oh; Park, Young Suk; Lim, Ho Yeong; Eltoukhy, Helmy; Kang, Won Ki; Lee, Woo Yong

    2015-01-01

    Sequencing of the mutant allele fraction of circulating cell-free DNA (cfDNA) derived from tumors is increasingly utilized to detect actionable genomic alterations in cancer. We conducted a prospective blinded study of a comprehensive cfDNA sequencing panel with 54 cancer genes. To evaluate the concordance between cfDNA and tumor DNA (tDNA), sequencing results were compared between cfDNA from plasma and genomic tumor DNA (tDNA). Utilizing next generation digital sequencing technology (DST), w...

  6. Mapping of the vaccinia virus DNA polymerase gene by marker rescue and cell-free translation of selected RNA

    International Nuclear Information System (INIS)

    The previous demonstration that a phosphonoacetate (PAA)-resistant (PAA/sup r/) vaccinia virus mutant synthesized an altered DNA polymerase provided the key to mapping this gene. Marker rescue was performed in cells infected with wild-type PAA-sensitive (PAA/sup s/) vaccinia by transfecting with calcium phosphate-precipitated DNA from a PAA/sup r/ mutant virus. Formation of PAA/sup r/ recombinants was measured by plaque assay in the presence of PAA. Of the 12 HindIII fragments cloned in plasmid or cosmid vectors, only fragment E conferred the PAA/sup r/ phenotype. Successive subcloning of the 15-kilobase HindIII fragment E localized the marker within a 7.5-kilobase BamHI-HindIII fragment and then within a 2.9-kilobase EcoRI fragment. The location of the DNA polymerase gene, about 57 kilobases from the left end of the genome, was confirmed by cell-free translation of mRNA selected by hybridization to plasmids containing regions of PAA/sup r/ vaccinia DNA active in marker rescue. A 100,000-dalton polypeptide that comigrated with authentic DNA polymerase was synthesized. Correspondence of the in vitro translation product with purified vaccinia DNA polymerase was established by peptide mapping

  7. SNPase-ARMS qPCR: Ultrasensitive Mutation-Based Detection of Cell-Free Tumor DNA in Melanoma Patients

    Science.gov (United States)

    Stadler, Julia; Eder, Johanna; Pratscher, Barbara; Brandt, Sabine; Schneller, Doris; Müllegger, Robert; Vogl, Claus; Trautinger, Franz; Brem, Gottfried; Burgstaller, Joerg P.

    2015-01-01

    Cell-free circulating tumor DNA in the plasma of cancer patients has become a common point of interest as indicator of therapy options and treatment response in clinical cancer research. Especially patient- and tumor-specific single nucleotide variants that accurately distinguish tumor DNA from wild type DNA are promising targets. The reliable detection and quantification of these single-base DNA variants is technically challenging. Currently, a variety of techniques is applied, with no apparent “gold standard”. Here we present a novel qPCR protocol that meets the conditions of extreme sensitivity and specificity that are required for detection and quantification of tumor DNA. By consecutive application of two polymerases, one of them designed for extreme base-specificity, the method reaches unprecedented sensitivity and specificity. Three qPCR assays were tested with spike-in experiments, specific for point mutations BRAF V600E, PTEN T167A and NRAS Q61L of melanoma cell lines. It was possible to detect down to one copy of tumor DNA per reaction (Poisson distribution), at a background of up to 200 000 wild type DNAs. To prove its clinical applicability, the method was successfully tested on a small cohort of BRAF V600E positive melanoma patients. PMID:26562020

  8. SNPase-ARMS qPCR: Ultrasensitive Mutation-Based Detection of Cell-Free Tumor DNA in Melanoma Patients.

    Directory of Open Access Journals (Sweden)

    Julia Stadler

    Full Text Available Cell-free circulating tumor DNA in the plasma of cancer patients has become a common point of interest as indicator of therapy options and treatment response in clinical cancer research. Especially patient- and tumor-specific single nucleotide variants that accurately distinguish tumor DNA from wild type DNA are promising targets. The reliable detection and quantification of these single-base DNA variants is technically challenging. Currently, a variety of techniques is applied, with no apparent "gold standard". Here we present a novel qPCR protocol that meets the conditions of extreme sensitivity and specificity that are required for detection and quantification of tumor DNA. By consecutive application of two polymerases, one of them designed for extreme base-specificity, the method reaches unprecedented sensitivity and specificity. Three qPCR assays were tested with spike-in experiments, specific for point mutations BRAF V600E, PTEN T167A and NRAS Q61L of melanoma cell lines. It was possible to detect down to one copy of tumor DNA per reaction (Poisson distribution, at a background of up to 200 000 wild type DNAs. To prove its clinical applicability, the method was successfully tested on a small cohort of BRAF V600E positive melanoma patients.

  9. Protein synthesis directly from PCR: progress and applications of cell-free protein synthesis with linear DNA.

    Science.gov (United States)

    Schinn, Song-Min; Broadbent, Andrew; Bradley, William T; Bundy, Bradley C

    2016-06-25

    A rapid, versatile method of protein expression and screening can greatly facilitate the future development of therapeutic biologics, proteomic drug targets and biocatalysts. An attractive candidate is cell-free protein synthesis (CFPS), a cell-lysate-based in vitro expression system, which can utilize linear DNA as expression templates, bypassing time-consuming cloning steps of plasmid-based methods. Traditionally, such linear DNA expression templates (LET) have been vulnerable to degradation by nucleases present in the cell lysate, leading to lower yields. This challenge has been significantly addressed in the recent past, propelling LET-based CFPS as a useful tool for studying, screening and engineering proteins in a high-throughput manner. Currently, LET-based CFPS has promise in fields such as functional proteomics, protein microarrays, and the optimization of complex biological systems. PMID:27085957

  10. Counting molecules in cell-free DNA and single cells RNA

    OpenAIRE

    Karlsson, Kasper

    2016-01-01

    The field of Molecular Biology got started in earnest with the discovery of the molecular structure of DNA. This lead to a surge of interest into the relationships between DNA, RNA and proteins, and to the development of fundamental tools for manipulating those substances, such as cutting, ligating, amplifying, visualizing and size-selecting DNA. With these tools at hand it was possible to begin sequencing DNA, a process that took a leap forward in 2005 with the advent of Next Generation Sequ...

  11. ACMG statement on noninvasive prenatal screening for fetal aneuploidy.

    Science.gov (United States)

    Gregg, Anthony R; Gross, S J; Best, R G; Monaghan, K G; Bajaj, K; Skotko, B G; Thompson, B H; Watson, M S

    2013-05-01

    Noninvasive assessment of the fetal genome is now possible using next-generation sequencing technologies. The isolation of fetal DNA fragments from maternal circulation in sufficient quantity and sizes, together with proprietary bioinformatics tools, now allows patients the option of noninvasive fetal aneuploidy screening. However, obstetric care providers must become familiar with the advantages and disadvantages of the utilization of this approach as analysis of cell-free fetal DNA moves into clinical practice. Once informed, clinicians can provide efficient pretest and posttest counseling with the goal of avoiding patient harm. It is in the public's best interest that test results contain key elements and that laboratories adhere to established quality control and proficiency testing standards. The analysis of cell-free fetal DNA in maternal circulation for fetal aneuploidy screening is likely the first of major steps toward the eventual application of whole fetal genome/whole fetal exome sequencing. PMID:23558255

  12. Noninvasive fetal genotyping of paternally inherited alleles

    NARCIS (Netherlands)

    Scheffer, P.G.

    2012-01-01

    The results presented in this thesis indicate that cell-free fetal DNA is a reliably analyte for prenatal genetic diagnosis in everyday clinical practice. Already, invasive procedures solely to determine the fetal sex or red cell antigen status belong to the past. Large-scale nation-wide prenatal sc

  13. Actionable mutations in plasma cell-free DNA in patients with advanced cancers referred for experimental targeted therapies

    Science.gov (United States)

    Janku, Filip; Angenendt, Philipp; Tsimberidou, Apostolia M.; Fu, Siqing; Naing, Aung; Falchook, Gerald S.; Hong, David S.; Holley, Veronica R.; Cabrilo, Goran; Wheler, Jennifer J.; Piha-Paul, Sarina A.; Zinner, Ralph G.; Bedikian, Agop Y.; Overman, Michael J.; Kee, Bryan K.; Kim, Kevin B.; Kopetz, E. Scott; Luthra, Rajyalakshmi; Diehl, Frank; Meric-Bernstam, Funda; Kurzrock, Razelle

    2015-01-01

    Cell-free (cf) DNA in the plasma of cancer patients offers an easily obtainable source of biologic material for mutation analysis. Plasma samples from 157 patients with advanced cancers who progressed on systemic therapy were tested for 21 mutations in BRAF, EGFR, KRAS, and PIK3CA using the BEAMing method and results were compared to mutation analysis of archival tumor tissue from a CLIA-certified laboratory obtained as standard of care from diagnostic or therapeutic procedures. Results were concordant for archival tissue and plasma cfDNA in 91% cases for BRAF mutations (kappa = 0.75, 95% confidence interval [CI] 0.63 – 0.88), in 99% cases for EGFR mutations (kappa = 0.90, 95% CI 0.71– 1.00), in 83% cases for KRAS mutations (kappa = 0.67, 95% CI 0.54 – 0.80) and in 91% cases for PIK3CA mutations (kappa = 0.65, 95% CI 0.46 – 0.85). Patients (n = 41) with > 1% of KRAS mutant cfDNA had a shorter median survival compared to 20 patients with 1% of mutant cfDNA (BRAF, EGFR, KRAS, or PIK3CA) had a shorter median survival compared to 33 patients with DNA (5.5 vs. 9.8 months, p = 0.001), which was confirmed in multivariable analysis. PMID:25980577

  14. The Most Favourable Procedure for the Isolation of Cell-free DNA from the Plasma of Iso-immunized RHD-negative Pregnant Women

    OpenAIRE

    Riyaz Ahmad Rather; Subhas Chandra Saha; Veena Dhawan

    2015-01-01

    Background: The ability to achieve quality recovery of cell- free foetal DNA is important for making non-invasive prenatal diagnoses. In this study, we performed quantita‐ tive and qualitative analyses of isolated DNA from mater‐ nal plasma, using different DNA-isolation methods. Method: DNA was isolated from 30 iso-immunized women via the QIAamp column-based method, using four differ‐ ent elution volumes and two conventionally based meth‐ ods. Real-time polymerase chain-reaction quantific...

  15. Circulating Cell Free DNA as the Diagnostic Marker for Ovarian Cancer: A Systematic Review and Meta-Analysis.

    Directory of Open Access Journals (Sweden)

    Quan Zhou

    Full Text Available Quantitative analyses of circulating cell-free DNA (cfDNA are potential methods for the detection of ovarian cancer. Many studies have evaluated these approaches, but the results were too inconsistent to be conclusive. This study is the first to systematically evaluate the accuracy of circulating cfDNA for the diagnosis of ovarian cancer by conducting meta-analysis.We searched PubMed, Embase, Cochrane Library and the Chinese National Knowledge Infrastructure (CNKI databases systematically for relevant literatures up to December 10, 2015. All analyses were conducted using Meta-DiSc1.4 and Stata 12.0 software. Sensitivity, specificity and other measures of accuracy of circulating cfDNA for the diagnosis of ovarian cancer were pooled. Meta-regression was performed to identify the sources of heterogeneity.This meta-analysis included a total of 9 studies, including 462 ovarian cancer patients and 407 controls. The summary estimates for quantitative analysis of circulating cfDNA in ovarian cancer screen were as follows: sensitivity, 0.70 (95% confidence interval (CI, 0.65-0.74; specificity, 0.90 (95% CI, 0.87-0.93; positive likelihood ratio, 6.60 (95% CI, 3.90-11.17; negative likelihood ratio, 0.34 (95% CI, 0.25-0.47; diagnostic odds ratio, 26.05 (95% CI, 14.67-46.26; and area under the curve, 0.89 (95% CI, 0.83-0.95, respectively. There was no statistical significance for the evaluation of publication bias.Current evidence suggests that quantitative analysis of cfDNA has unsatisfactory sensitivity but acceptable specificity for the diagnosis of ovarian cancer. Further large-scale prospective studies are required to validate the potential applicability of using circulating cfDNA alone or in combination with conventional markers as diagnostic biomarker for ovarian cancer and explore potential factors that may influence the accuracy of ovarian cancer diagnosis.

  16. Posttest risk calculation following positive noninvasive prenatal screening using cell-free DNA in maternal plasma.

    Science.gov (United States)

    Benn, Peter

    2016-06-01

    Noninvasive prenatal screening (NIPS) for fetal chromosome defects has high sensitivity and specificity but is not fully diagnostic. In response to a desire to provide more information to individual women with positive NIPS results, 2 online calculators have been developed to calculate posttest risk (PTR). Use of these calculators is critically reviewed. There is a mathematically dictated requirement for a precise estimate for the specificity to provide an accurate PTR. This is illustrated by showing that a 0.1% decrease in the value for specificities for trisomies 21, 18, and 13 can reduce the PTR from 79-64% for trisomy 21, 39-27% for trisomy 18, and 21-13% for trisomy 13, respectively. Use of the calculators assumes that sensitivity and specificity are constant for all women receiving the test but there is evidence that discordancy between screening results and true fetal karyotype is more common for older women. Use of an appropriate value for the prior risk is also important and for rare disorders there is considerable uncertainty regarding prevalence. For example, commonly used rates for trisomy 13, monosomy-X, triploidy, and 22q11.2 deletion syndrome can vary by >4-fold and this can translate into large differences in PTR. When screening for rare disorders, it may not be possible to provide a reliable PTR if there is uncertainty over the false-positive rate and/or prevalence. These limitations, per se, do not negate the value of screening for rare conditions. However, counselors need to carefully weigh the validity of PTR before presenting them to patients. Additional epidemiologic and NIPS outcome data are needed. PMID:26772793

  17. Cell-free assay measuring repair DNA synthesis in human fibroblasts

    International Nuclear Information System (INIS)

    Osmotic disruption of confluent cultured human fibroblasts that have been irradiated or exposed to chemical carcinogens allows the specific measurement of repair DNA synthesis using dTTP as a precursor. Fibroblasts similarly prepared from various xeroderma pigmentosum cell lines show the deficiencies of uv-induced DNA synthesis predicted from in vivo studies, while giving normal responses to methylmethanesulfonate. A pyrimidine-dimer-specific enzyme, T4 endonuclease V, stimulated the rate of uv-induced repair synthesis with normal and xeroderma pigmentosum cell lines. This system should prove useful for identifying agents that induce DNA repair, and cells that respond abnormally to such induction. It should also be applicable to an in vitro complementation assay with repair-defective cells and proteins obtained from repair-proficient cells. Finally, by using actively growing fibroblasts and thymidine in the system, DNA replication can be measured and studied in vitro

  18. Diagnostic and prognostic value of cell free circulating Schistosoma mansoni DNA: an experimental study.

    Science.gov (United States)

    Eraky, Maysa Ahmad; Aly, Nagwa Shaban Mohamed

    2016-09-01

    Searching for a more sensitive and accurate marker for schistosomiasis diagnosis and treatment follow up is a potential necessity. Hereby, we evaluated usefulness of circulating free DNA as a marker for schistosomiasis diagnosis, assessing drug efficacy and monitoring the control interventions impact using SYBR green real-time PCR. A batch of mice were infected by 90 ± 10 Schistosoma mansoni cercariae. Starting from the 2nd day post infection (p.i.), groups of 2 or 3 mice were sacrificed every 3 days until 30 days p.i. The remaining animals were treated by a single dose of 400 mg/kg mefloquine and sacrificed in group at 5, 10, 21 days post treatment (35, 40, 51 days p.i.). Using SYBR green real time qPCR, pooled sera DNA were extracted and amplified. The results showed that, circulating free S. mansoni DNA was detected from the 2nd day post infection (p.i.) onwards with gradual decrease in the cycle threshold value Ct which indicates the gradual elevation of the DNA level (Log quantity was 2.6-3.1 IU/ml), As the infection progressed, DNA quantity was increased(Log quantity was 6.29 IU/ml). Initial increase of circulating free DNA was observed 10 days post treatment (40 days p.i.) (Log quantity was 7.38 IU/ml). That was followed by a progressive decrease in DNA level by the end of 21st day, post treatment (51 p.i.) (Log quantity 4.35 IU/ml). In conclusion, circulating free S. mansoni DNA is a reliable marker in the diagnosis of schistosomiasis and for assessing drug efficacy and monitoring the impact of control interventions. PMID:27605830

  19. Fatal outcome in bacteremia is characterized by high plasma cell free DNA concentration and apoptotic DNA fragmentation: a prospective cohort study.

    Directory of Open Access Journals (Sweden)

    Reetta Huttunen

    Full Text Available INTRODUCTION: Recent studies have shown that apoptosis plays a critical role in the pathogenesis of sepsis. High plasma cell free DNA (cf-DNA concentrations have been shown to be associated with sepsis outcome. The origin of cf-DNA is unclear. METHODS: Total plasma cf-DNA was quantified directly in plasma and the amplifiable cf-DNA assessed using quantitative PCR in 132 patients with bacteremia caused by Staphylococcus aureus, Streptococcus pneumoniae, ß-hemolytic streptococcae or Escherichia coli. The quality of cf-DNA was analyzed with a DNA Chip assay performed on 8 survivors and 8 nonsurvivors. Values were measured on days 1-4 after positive blood culture, on day 5-17 and on recovery. RESULTS: The maximum cf-DNA values on days 1-4 (n = 132 were markedly higher in nonsurvivors compared to survivors (2.03 vs 1.26 ug/ml, p1.52 ug/ml remained an independent risk factor for case fatality in a logistic regression model. Qualitative analysis of cf-DNA showed that cf-DNA displayed a predominating low-molecular-weight cf-DNA band (150-200 bp in nonsurvivors, corresponding to the size of the apoptotic nucleosomal DNA. cf-DNA concentration showed a significant positive correlation with visually graded apoptotic band intensity (R = 0.822, p<0.001. CONCLUSIONS: Plasma cf-DNA concentration proved to be a specific independent prognostic biomarker in bacteremia. cf-DNA displayed a predominating low-molecular-weight cf-DNA band in nonsurvivors corresponding to the size of apoptotic nucleosomal DNA.

  20. High cell-free DNA predicts fatal outcome among Staphylococcus aureus bacteraemia patients with intensive care unit treatment.

    Directory of Open Access Journals (Sweden)

    Erik Forsblom

    Full Text Available INTRODUCTION: Among patients with bacteraemia or sepsis the plasma cell-free DNA (cf-DNA biomarker has prognostic value and Pitt bacteraemia scores predict outcome. We evaluated the prognostic value of plasma cf-DNA in patients with Staphylococcus aureus bacteraemia (SAB treated in the ICU or in the general ward. METHODS: 418 adult patients with positive blood culture for S. aureus were prospectively followed for 90 days. SAB patients were grouped according to ICU treatment: 99 patients were treated in ICU within 7 days of documented SAB whereas 319 patients were managed outside ICU. Pitt bacteraemia scores were assessed at hospital arrival and cf-DNA was measured at days 3 and 5 from positive blood culture. RESULTS: SAB patients with high Pitt bacteraemia scores and ICU treatment presented higher cf-DNA values as compared to SAB patients with low Pitt bacteraemia scores and non-ICU treatment at both days 3 and 5. Among ICU patients cf-DNA >1.99 µg/ml at day 3 predicted death with a sensitivity of 67% and a specificity of 77% and had an AUC in receiver operating characteristic analysis of 0.71 (p1.99 µg/ml value demonstrated a strong association to high Pitt bacteraemia scores (≥ 4 points (p<0.000. After controlling for all prognostic markers, Pitt bacteraemia scores ≥ 4 points at hospital admission (OR 4.47, p<0.000 and day 3 cf-DNA (OR 3.56, p<0.001 were the strongest factors significantly predicting outcome in ICU patients. cf-DNA at day 5 did not predict fatal outcome. CONCLUSION: High cf-DNA concentrations were observed among patients with high Pitt bacteraemia scores and ICU treatment. Pitt bacteraemia scores (≥ 4 points and cf-DNA at day 3 from positive blood culture predicted death among SAB patients in ICU and were found to be independent prognostic markers. cf-DNA had no prognostic value among non-ICU patients.

  1. Cell Free DNA of Tumor Origin Induces a 'Metastatic' Expression Profile in HT-29 Cancer Cell Line.

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    István Fűri

    Full Text Available Epithelial cells in malignant conditions release DNA into the extracellular compartment. Cell free DNA of tumor origin may act as a ligand of DNA sensing mechanisms and mediate changes in epithelial-stromal interactions.To evaluate and compare the potential autocrine and paracrine regulatory effect of normal and malignant epithelial cell-related DNA on TLR9 and STING mediated pathways in HT-29 human colorectal adenocarcinoma cells and normal fibroblasts.DNA isolated from normal and tumorous colonic epithelia of fresh frozen surgically removed tissue samples was used for 24 and 6 hour treatment of HT-29 colon carcinoma and HDF-α fibroblast cells. Whole genome mRNA expression analysis and qRT-PCR was performed for the elements/members of TLR9 signaling pathway. Immunocytochemistry was performed for epithelial markers (i.e. CK20 and E-cadherin, DNA methyltransferase 3a (DNMT3a and NFκB (for treated HDFα cells.Administration of tumor derived DNA on HT29 cells resulted in significant (p<0.05 mRNA level alteration in 118 genes (logFc≥1, p≤0.05, including overexpression of metallothionein genes (i.e. MT1H, MT1X, MT1P2, MT2A, metastasis-associated genes (i.e. TACSTD2, MACC1, MALAT1, tumor biomarker (CEACAM5, metabolic genes (i.e. INSIG1, LIPG, messenger molecule genes (i.e. DAPP, CREB3L2. Increased protein levels of CK20, E-cadherin, and DNMT3a was observed after tumor DNA treatment in HT-29 cells. Healthy DNA treatment affected mRNA expression of 613 genes (logFc≥1, p≤0.05, including increased expression of key adaptor molecules of TLR9 pathway (e.g. MYD88, IRAK2, NFκB, IL8, IL-1β, STING pathway (ADAR, IRF7, CXCL10, CASP1 and the FGF2 gene.DNA from tumorous colon epithelium, but not from the normal epithelial cells acts as a pro-metastatic factor to HT-29 cells through the overexpression of pro-metastatic genes through TLR9/MYD88 independent pathway. In contrast, DNA derived from healthy colonic epithelium induced TLR9 and STING signaling

  2. Applications for quantitative measurement of BRAF V600 mutant cell-free tumor DNA in the plasma of patients with metastatic melanoma.

    Science.gov (United States)

    Schreuer, Max; Meersseman, Geert; van Den Herrewegen, Sari; Jansen, Yanina; Seremet, Teofila; Bott, Ambre; Chevolet, Ines; Wilgenhof, Sofie; Maertens, Geert; Neyns, Bart

    2016-04-01

    Small fragments of cell-free DNA that are shed by normal and tumor cells can be detected in the plasma of patients with advanced melanoma. Quantitative measurement of BRAF V600 mutant DNA within the cell-free DNA holds promise as a tumor-specific biomarker for diagnosis and therapeutic monitoring in patients with BRAF V600 mutant melanoma. Allele-specific quantitative PCR analysis for BRAF V600 E/E2/D/K/R/M mutations on DNA extracted from 1 ml of plasma is currently under evaluation in a number of ongoing prospective clinical studies. We report five patient cases that indicate the potential applications and utility of quantitative measurements of BRAF V600 mutant cell-free tumor DNA as a diagnostic test and as a therapeutic monitoring tool in stage IV melanoma patients treated with BRAF-targeted therapy or immunotherapy. Finally, we offer novel insights into the dynamics of cell-free tumor DNA in melanoma. PMID:26636909

  3. Cell-free DNA in Human Follicular Microenvironment: New Prognostic Biomarker to Predict in vitro Fertilization Outcomes.

    Directory of Open Access Journals (Sweden)

    Sabine Traver

    Full Text Available Cell-free DNA (cfDNA fragments, detected in blood and in other biological fluids, are released from apoptotic and/or necrotic cells. CfDNA is currently used as biomarker for the detection of many diseases such as some cancers and gynecological and obstetrics disorders. In this study, we investigated if cfDNA levels in follicular fluid (FF samples from in vitro fertilization (IVF patients, could be related to their ovarian reserve status, controlled ovarian stimulation (COS protocols and IVF outcomes. Therefore, 117 FF samples were collected from women (n = 117 undergoing IVF/Intra-cytoplasmic sperm injection (ICSI procedure and cfDNA concentration was quantified by ALU-quantitative PCR. We found that cfDNA level was significantly higher in FF samples from patients with ovarian reserve disorders (low functional ovarian reserve or polycystic ovary syndrome than from patients with normal ovarian reserve (2.7 ± 2.7 ng/μl versus 1.7 ± 2.3 ng/μl, respectively, p = 0.03. Likewise, FF cfDNA levels were significant more elevated in women who received long ovarian stimulation (> 10 days or high total dose of gonadotropins (≥ 3000 IU/l than in women who received short stimulation duration (7-10 days or total dose of gonadotropins < 3000 IU/l (2.4 ± 2.8 ng/μl versus 1.5 ± 1.9 ng/μl, p = 0.008; 2.2 ± 2.3 ng/μl versus 1.5 ± 2.1 ng/μl, p = 0.01, respectively. Finally, FF cfDNA level was an independent and significant predictive factor for pregnancy outcome (adjusted odds ratio = 0.69 [0.5; 0.96], p = 0.03. In multivariate analysis, the Receiving Operator Curve (ROC analysis showed that the performance of FF cfDNA in predicting clinical pregnancy reached 0.73 [0.66-0.87] with 88% specificity and 60% sensitivity. CfDNA might constitute a promising biomarker of follicular micro-environment quality which could be used to predict IVF prognosis and to enhance female infertility management.

  4. Cell-free DNA in Human Follicular Microenvironment: New Prognostic Biomarker to Predict in vitro Fertilization Outcomes

    Science.gov (United States)

    Mullet, Tiffany; Molinari, Nicolas; Vincens, Claire; Anahory, Tal; Hamamah, Samir

    2015-01-01

    Cell-free DNA (cfDNA) fragments, detected in blood and in other biological fluids, are released from apoptotic and/or necrotic cells. CfDNA is currently used as biomarker for the detection of many diseases such as some cancers and gynecological and obstetrics disorders. In this study, we investigated if cfDNA levels in follicular fluid (FF) samples from in vitro fertilization (IVF) patients, could be related to their ovarian reserve status, controlled ovarian stimulation (COS) protocols and IVF outcomes. Therefore, 117 FF samples were collected from women (n = 117) undergoing IVF/Intra-cytoplasmic sperm injection (ICSI) procedure and cfDNA concentration was quantified by ALU-quantitative PCR. We found that cfDNA level was significantly higher in FF samples from patients with ovarian reserve disorders (low functional ovarian reserve or polycystic ovary syndrome) than from patients with normal ovarian reserve (2.7 ± 2.7 ng/μl versus 1.7 ± 2.3 ng/μl, respectively, p = 0.03). Likewise, FF cfDNA levels were significant more elevated in women who received long ovarian stimulation (> 10 days) or high total dose of gonadotropins (≥ 3000 IU/l) than in women who received short stimulation duration (7–10 days) or total dose of gonadotropins < 3000 IU/l (2.4 ± 2.8 ng/μl versus 1.5 ± 1.9 ng/μl, p = 0.008; 2.2 ± 2.3 ng/μl versus 1.5 ± 2.1 ng/μl, p = 0.01, respectively). Finally, FF cfDNA level was an independent and significant predictive factor for pregnancy outcome (adjusted odds ratio = 0.69 [0.5; 0.96], p = 0.03). In multivariate analysis, the Receiving Operator Curve (ROC) analysis showed that the performance of FF cfDNA in predicting clinical pregnancy reached 0.73 [0.66–0.87] with 88% specificity and 60% sensitivity. CfDNA might constitute a promising biomarker of follicular micro-environment quality which could be used to predict IVF prognosis and to enhance female infertility management. PMID:26288130

  5. Circulating cell free DNA as a predictor of systemic lupus erythematosus severity and monitoring of therapy

    Directory of Open Access Journals (Sweden)

    Olfat M. Hendy

    2016-01-01

    Conclusion: Our findings support that the measurement of cf-DNA appears to be a useful marker in addition to laboratory tests used in SLE diagnosis. High correlation with markers of disease severity suggesting its role in disease pathogenesis and decreasing its level after therapy makes it to be a marker of treatment follow-up.

  6. Cell free DNA testing-interpretation of results using an online calculator.

    Science.gov (United States)

    Grace, Matthew R; Hardisty, Emily; Green, Noah S; Davidson, Emily; Stuebe, Alison M; Vora, Neeta L

    2015-07-01

    All pregnant women, regardless of age, should be offered screening or invasive testing for chromosomal abnormalities at advertise and report a test's sensitivity and specificity as a means to describe the test's accuracy. The positive predictive value (PPV) of a screening test (the proportion of positive results that are truly positive) is a function of the prevalence of the condition in a population and often is not reported in direct-to-patient advertising. False-positive cfDNA screening tests have been reported, and there is evidence that some women are deciding to terminate their pregnancy without confirmatory testing. We believe that laboratories should disclose the patient-specific PPV of cfDNA screening for aneuploidy on result reports. To assist with counseling patients about the benefits, risks, and limitations of aneuploidy screening with the use of cfDNA and to demonstrate the relationship between an a priori risk and PPV, we developed a web-based calculator to estimate the PPV of the 4 commercially available cfDNA testing platforms for which data have been published. Estimates are made with the use of a patient's age and gestational age-related risk of trisomy 21, 18 and 13 or an a priori risk that is based on other findings. This web-based calculator is an aid for providers and genetic counselors to illustrate the relationship between disease prevalence and a test's PPV. It has enhanced our counseling of patients both before they elect noninvasive prenatal screening and after they receive a positive result. PMID:25957020

  7. Urine Cell-Free DNA Integrity Analysis for Early Detection of Prostate Cancer Patients

    OpenAIRE

    Samanta Salvi; Giorgia Gurioli; Filippo Martignano; Flavia Foca; Roberta Gunelli; Giacomo Cicchetti; Ugo De Giorgi; Wainer Zoli; Daniele Calistri; Valentina Casadio

    2015-01-01

    Introduction. The detection of tumor-specific markers in urine has paved the way for new early noninvasive diagnostic approaches for prostate cancer. We evaluated the DNA integrity in urine supernatant to verify its capacity to discriminate between prostate cancer and benign diseases of the urogenital tract. Patients and Methods. A total of 131 individuals were enrolled: 67 prostate cancer patients and 64 patients with benign diseases of the urogenital tract (control group). Prostate-specific...

  8. Silver Nanoscale Hexagonal Column Chips for Detecting Cell-free DNA and Circulating Nucleosomes in Cancer Patients

    Science.gov (United States)

    Ito, Hiroaki; Hasegawa, Katsuyuki; Hasegawa, Yuuki; Nishimaki, Tadashi; Hosomichi, Kazuyoshi; Kimura, Satoshi; Ohba, Motoi; Yao, Hiroshi; Onimaru, Manabu; Inoue, Ituro; Inoue, Haruhiro

    2015-01-01

    Blood tests, which are commonly used for cancer screening, generally have low sensitivity. Here, we developed a novel rapid and simple method to generate silver nanoscale hexagonal columns (NHCs) for use in surface-enhanced Raman scattering (SERS). We reported that the intensity of SERS spectra of clinical serum samples obtained from gastrointestinal cancer patients is was significantly higher than that of SERS spectra of clinical serum samples obtained from non-cancer patients. We estimated the combined constituents on silver NHCs by using a field emission-type scanning electron microscope, Raman microscopes, and a 3D laser scanning confocal microscope. We obtained the Raman scattering spectra of samples of physically fractured cells and clinical serum. No spectra were obtained for chemically lysed cultured cells and DNA, RNA, and protein extracted from cultured cells. We believe that our method, which uses SERS with silver NHCs to detect circulating nucleosomes bound by methylated cell-free DNA, may be successfully implemented in blood tests for cancer screening. PMID:25994878

  9. Circulating Cell Free DNA as the Diagnostic Marker for Ovarian Cancer: A Systematic Review and Meta-Analysis

    Science.gov (United States)

    Leng, Bingjie; Zheng, Wenfei; He, Ze; Zuo, Manzhen; Chen, Aihua

    2016-01-01

    Background Quantitative analyses of circulating cell-free DNA (cfDNA) are potential methods for the detection of ovarian cancer. Many studies have evaluated these approaches, but the results were too inconsistent to be conclusive. This study is the first to systematically evaluate the accuracy of circulating cfDNA for the diagnosis of ovarian cancer by conducting meta-analysis. Methods We searched PubMed, Embase, Cochrane Library and the Chinese National Knowledge Infrastructure (CNKI) databases systematically for relevant literatures up to December 10, 2015. All analyses were conducted using Meta-DiSc1.4 and Stata 12.0 software. Sensitivity, specificity and other measures of accuracy of circulating cfDNA for the diagnosis of ovarian cancer were pooled. Meta-regression was performed to identify the sources of heterogeneity. Results This meta-analysis included a total of 9 studies, including 462 ovarian cancer patients and 407 controls. The summary estimates for quantitative analysis of circulating cfDNA in ovarian cancer screen were as follows: sensitivity, 0.70 (95% confidence interval (CI), 0.65–0.74); specificity, 0.90 (95% CI, 0.87–0.93); positive likelihood ratio, 6.60 (95% CI, 3.90–11.17); negative likelihood ratio, 0.34 (95% CI, 0.25–0.47); diagnostic odds ratio, 26.05 (95% CI, 14.67–46.26); and area under the curve, 0.89 (95% CI, 0.83–0.95), respectively. There was no statistical significance for the evaluation of publication bias. Conclusions Current evidence suggests that quantitative analysis of cfDNA has unsatisfactory sensitivity but acceptable specificity for the diagnosis of ovarian cancer. Further large-scale prospective studies are required to validate the potential applicability of using circulating cfDNA alone or in combination with conventional markers as diagnostic biomarker for ovarian cancer and explore potential factors that may influence the accuracy of ovarian cancer diagnosis. PMID:27253331

  10. Pre-analytical variables of circulating cell-free nucleosomes containing 5-methylcytosine DNA or histone modification H3K9Me3

    DEFF Research Database (Denmark)

    Rasmussen, Louise; Herzog, Marielle; Aastrup rømer, Eva Christine;

    2016-01-01

    AIM: To evaluate pre-analytical variables of circulating cell-free nucleosomes containing 5-methylcytosine DNA (5mC) or histone modification H3K9Me3 (H3K9Me3). MATERIALS AND METHODS: Six studies were designed to assess the possible influence of pre-analytical variables. Study 1: influence of stasis...

  11. Evaluation of INK4A promoter methylation using pyrosequencing and circulating cell-free DNA from patients with hepatocellular carcinoma

    Science.gov (United States)

    Kirk, Jason L.; Merwat, Shehzad N.; Ju, Hyunsu; Soloway, Roger D.; Wieck, Lucas R.; Li, Albert; Okorodudu, Anthony O.; Petersen, John R.; Abdulla, Nihal E.; Duchini, Andrea; Cicalese, Luca; Rastellini, Cristiana; Hu, Peter C.; Dong, Jianli

    2015-01-01

    Background Hyper-methylation of CpG dinucleotides in the promoter region of inhibitor of cyclin-dependent kinase 4A (INK4A) has been reported in 60%–80% of hepatocellular carcinoma (HCC). As INK4A promoter hypermethylation event occurs early in HCC progression, the quantification of INK4A promoter methylation in blood sample may represent a useful biomarker for non-invasive diagnosis and prediction of response to therapy. Methods We examined INK4A promoter methylation using circulating cell-free DNA (ccfDNA) in a total of 109 serum specimens, including 66 HCC and 43 benign chronic liver diseases. Methylation of the individual seven CpG sites was examined using pyrosequencing. Results Our results showed that there were significantly higher levels of methylated INK4A in HCC specimens than controls and that the seven CpG sites had different levels of methylation and might exist in different PCR amplicons. The area under receiver operating characteristic (ROC) curve was 0.82, with 65.3% sensitivity and 87.2% specificity at 5% (LOD), 39.0% sensitivity and 96.5% specificity at 7% LOD, and 20.3% sensitivity and 98.8% specificity at 10% LOD, respectively. Conclusions Our results support additional studies incorporating INK4A methylation testing of ccfDNA to further validate the diagnostic, predictive, and prognostic characteristics of this biomarker in HCC patients. The knowledge of the existence of epi-alleles should help improve assay design to maximize detection. PMID:24406287

  12. KRAS G12V Mutation Detection by Droplet Digital PCR in Circulating Cell-Free DNA of Colorectal Cancer Patients.

    Science.gov (United States)

    Olmedillas López, Susana; García-Olmo, Dolores C; García-Arranz, Mariano; Guadalajara, Héctor; Pastor, Carlos; García-Olmo, Damián

    2016-01-01

    KRAS mutations are responsible for resistance to anti-epidermal growth factor receptor (EGFR) therapy in colorectal cancer patients. These mutations sometimes appear once treatment has started. Detection of KRAS mutations in circulating cell-free DNA in plasma ("liquid biopsy") by droplet digital PCR (ddPCR) has emerged as a very sensitive and promising alternative to serial biopsies for disease monitoring. In this study, KRAS G12V mutation was analyzed by ddPCR in plasma DNA from 10 colorectal cancer patients and compared to six healthy donors. The percentage of KRAS G12V mutation relative to wild-type sequences in tumor-derived DNA was also determined. KRAS G12V mutation circulating in plasma was detected in 9 of 10 colorectal cancer patients whose tumors were also mutated. Colorectal cancer patients had 35.62 copies of mutated KRAS/mL plasma, whereas in healthy controls only residual copies were found (0.62 copies/mL, p = 0.0066). Interestingly, patients with metastatic disease showed a significantly higher number of mutant copies than M0 patients (126.25 versus 9.37 copies/mL, p = 0.0286). Wild-type KRAS was also significantly elevated in colorectal cancer patients compared to healthy controls (7718.8 versus 481.25 copies/mL, p = 0.0002). In conclusion, KRAS G12V mutation is detectable in plasma of colorectal cancer patients by ddPCR and could be used as a non-invasive biomarker. PMID:27043547

  13. KRAS G12V Mutation Detection by Droplet Digital PCR in Circulating Cell-Free DNA of Colorectal Cancer Patients

    Directory of Open Access Journals (Sweden)

    Susana Olmedillas López

    2016-04-01

    Full Text Available KRAS mutations are responsible for resistance to anti-epidermal growth factor receptor (EGFR therapy in colorectal cancer patients. These mutations sometimes appear once treatment has started. Detection of KRAS mutations in circulating cell-free DNA in plasma (“liquid biopsy” by droplet digital PCR (ddPCR has emerged as a very sensitive and promising alternative to serial biopsies for disease monitoring. In this study, KRAS G12V mutation was analyzed by ddPCR in plasma DNA from 10 colorectal cancer patients and compared to six healthy donors. The percentage of KRAS G12V mutation relative to wild-type sequences in tumor-derived DNA was also determined. KRAS G12V mutation circulating in plasma was detected in 9 of 10 colorectal cancer patients whose tumors were also mutated. Colorectal cancer patients had 35.62 copies of mutated KRAS/mL plasma, whereas in healthy controls only residual copies were found (0.62 copies/mL, p = 0.0066. Interestingly, patients with metastatic disease showed a significantly higher number of mutant copies than M0 patients (126.25 versus 9.37 copies/mL, p = 0.0286. Wild-type KRAS was also significantly elevated in colorectal cancer patients compared to healthy controls (7718.8 versus 481.25 copies/mL, p = 0.0002. In conclusion, KRAS G12V mutation is detectable in plasma of colorectal cancer patients by ddPCR and could be used as a non-invasive biomarker.

  14. Cell-free mitochondrial DNA in CSF is associated with early viral rebound, inflammation, and severity of neurocognitive deficits in HIV infection.

    Science.gov (United States)

    Pérez-Santiago, Josué; Schrier, Rachel D; de Oliveira, Michelli F; Gianella, Sara; Var, Susanna R; Day, Tyler R C; Ramirez-Gaona, Miguel; Suben, Jesse D; Murrell, Ben; Massanella, Marta; Cherner, Mariana; Smith, Davey M; Ellis, Ronald J; Letendre, Scott L; Mehta, Sanjay R

    2016-04-01

    Cell-free mitochondiral DNA (mtDNA) is an immunogenic molecule associated with many inflammatory conditions. We evaluated the relationship between cell-free mtDNA in cerebrospinal fluid (CSF) and neurocognitive performance and inflammation during HIV infection. In a cross-sectional analysis, we evaluated the association of mtDNA levels with clinical assessments, inflammatory markers, and neurocognitive performance in 28 HIV-infected individuals. In CSF, we measured mtDNA levels by droplet digital PCR, and soluble CD14 and CD163, neurofilament light, and neopterin by ELISA. In blood and CSF, we measured soluble IP-10, MCP-1, TNF-α, and IL-6 by ELISA, and intracellular expression of IL-2, IFN-γ, and TNF-α in CD4(+) and CD8(+) T cells by flow cytometry. We also evaluated the relationship between CSF pleocytosis and mtDNA longitudinally in another set of five individuals participating in an antiretroviral treatment (ART) interruption study. Cell-free CSF mtDNA levels strongly correlated with neurocognitive performance among individuals with neurocognitive impairment (NCI) (r = 0.77, p = 0.001). CSF mtDNA also correlated with levels of IP-10 in CSF (r = 0.70, p = 0.007) and MCP-1 in blood plasma (r = 0.66, p = 0.01) in individuals with NCI. There were no significant associations between inflammatory markers and mtDNA in subjects without NCI, and levels of mtDNA did not differ between subjects with and without NCI. MtDNA levels preceded pleocytosis and HIV RNA following ART interruption. Cell-free mtDNA in CSF was strongly associated with the severity of neurocognitive dysfunction and inflammation only in individuals with NCI. Our findings suggest that within a subset of subjects cell-free CSF mtDNA is associated with inflammation and degree of NCI. PMID:26428514

  15. Quantitative characterization of pyrimidine dimer excision from UV-irradiated DNA (excision capacity) by cell-free extracts of the yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Cell-free extracts from wild-type yeast (RAD+) and from rad mutants belonging to the RAD3 epistatic group (rad1-1, rad2-1, rad3-1, rad4-1) contain activities catalyzing the excision of pyrimidine dimers (PD) from purified ultraviolet-irradiated DNA which was not pre-treated with exogenous UV-endonuclease. The level of these activities in cell-free extracts from rad mutants did not differ from that in wild-type extract and was close to the in vivo excision capacity of the latter calculated from the LD37 (about 104 PD per haploid genome). (Auth.)

  16. Complementation of the xeroderma pigmentosum DNA repair synthesis defect with Escherichia coli UvrABC proteins in a cell-free system.

    OpenAIRE

    Hansson, J; Grossman, L; Lindahl, T; Wood, R D

    1990-01-01

    A newly developed cell-free system was used to study DNA repair synthesis carried out by extracts from human cell lines in vitro. Extracts from a normal human lymphoid cell line and from cell lines established from individuals with hereditary dysplastic nevus syndrome perform damage-dependent repair synthesis in plasmid DNA treated with cis- or trans-diamminedichloro-platinum(II) or irradiated with ultraviolet light. Cell extracts of xeroderma pigmentosum origin (complementation groups A, C, ...

  17. Analytical and Clinical Validation of a Digital Sequencing Panel for Quantitative, Highly Accurate Evaluation of Cell-Free Circulating Tumor DNA

    OpenAIRE

    Lanman, Richard B.; Mortimer, Stefanie A.; Zill, Oliver A.; Sebisanovic, Dragan; Lopez, Rene; Blau, Sibel; Collisson, Eric A.; Divers, Stephen G.; Hoon, Dave S.B.; Kopetz, E. Scott; Lee, Jeeyun; Nikolinakos, Petros G.; Baca, Arthur M.; Kermani, Bahram G.; Eltoukhy, Helmy

    2015-01-01

    Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital SequencingTM is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-relate...

  18. Circulating cell-free methylated DNA and lactate dehydrogenase release in colorectal cancer

    International Nuclear Information System (INIS)

    Hypermethylation of DNA is an epigenetic alteration commonly found in colorectal cancer (CRC) and can also be detected in blood samples of cancer patients. Methylation of the genes helicase-like transcription factor (HLTF) and hyperplastic polyposis 1 (HPP1) have been proposed as prognostic, and neurogenin 1 (NEUROG1) as diagnostic biomarker. However the underlying mechanisms leading to the release of these genes are unclear. This study aimed at examining the possible correlation of the presence of methylated genes NEUROG1, HLTF and HPP1 in serum with tissue breakdown as a possible mechanism using serum lactate dehydrogenase (LDH) as a surrogate marker. Additionally the prognostic impact of these markers was examined. Pretherapeutic serum samples from 259 patients from all cancer stages were analyzed. Presence of hypermethylation of the genes HLTF, HPP1, and NEUROG1 was examined using methylation-specific quantitative PCR (MethyLight). LDH was determined using an UV kinetic test. Hypermethylation of HLTF and HPP1 was detected significantly more often in patients with elevated LDH levels (32% vs. 12% [p = 0.0005], and 68% vs. 11% [p < 0.0001], respectively). Also, higher LDH values correlated with a higher percentage of a fully methylated reference in a linear fashion (Spearman correlation coefficient 0.18 for HLTF [p = 0.004]; 0.49 [p < .0001] for HPP1). No correlation between methylation of NEUROG1 and LDH was found in this study. Concerning the clinical characteristics, high levels of LDH as well as methylation of HLTF and HPP1 were significantly associated with larger and more advanced stages of CRC. Accordingly, these three markers were correlated with significantly shorter survival in the overall population. Moreover, all three identified patients with a worse prognosis in the subgroup of stage IV patients. We were able to provide evidence that methylation of HLTF and especially HPP1 detected in serum is strongly correlated with cell death in CRC using LDH as

  19. Admission cell free DNA levels predict 28-day mortality in patients with severe sepsis in intensive care.

    Directory of Open Access Journals (Sweden)

    Avital Avriel

    Full Text Available The aim of the current study is to assess the mortality prediction accuracy of circulating cell-free DNA (CFD level at admission measured by a new simplified method.CFD levels were measured by a direct fluorescence assay in severe sepsis patients on intensive care unit (ICU admission. In-hospital and/or twenty eight day all-cause mortality was the primary outcome.Out of 108 patients with median APACHE II of 20, 32.4% have died in hospital/or at 28-day. CFD levels were higher in decedents: median 3469.0 vs. 1659 ng/ml, p<0.001. In multivariable model APACHE II score and CFD (quartiles were significantly associated with the mortality: odds ratio of 1.05, p = 0.049 and 2.57, p<0.001 per quartile respectively. C-statistics for the models was 0.79 for CFD and 0.68 for APACHE II. Integrated discrimination improvement (IDI analyses showed that CFD and CFD+APACHE II score models had better discriminatory ability than APACHE II score alone.CFD level assessed by a new, simple fluorometric-assay is an accurate predictor of acute mortality among ICU patients with severe sepsis. Comparison of CFD to APACHE II score and Procalcitonin (PCT, suggests that CFD has the potential to improve clinical decision making.

  20. Increased serum cell-free DNA levels in relation to inflammation are predictive of distant metastasis of esophageal squamous cell carcinoma

    OpenAIRE

    TOMOCHIKA, SHINOBU; Iizuka, Norio; Watanabe, Yusaku; TSUTSUI, MASAHITO; Takeda, Shigeru; Yoshino, Shigefumi; ICHIHARA, KIYOSHI; Oka, Masaaki

    2010-01-01

    Distant metastasis hinders a favorable outcome for patients with esophageal squamous cell carcinoma (ESCC) by limiting the surgical cure. The levels of cell-free DNA (cfDNA) in the blood have served as a predictor for metastasis and recurrence in distant organs in liver cancer. Thus, this study tested the clinical efficacy of serum cfDNA levels as a predictive marker for distant metastasis of ESCC. We investigated cfDNA levels in a cohort of 101 ESCC patients and 46 age- and gender-matched co...

  1. The severity of alpha-particle-induced DNA damage is revealed by exposure to cell-free extracts

    International Nuclear Information System (INIS)

    The rejoining of single-strand breaks induced by α-particle and γ irradiation in plasmid DNA under two scavenging conditions has been compared. At the two scavenger conditions has been compared. At the two scavenger capacities used of 1.5 x 107 and 3 x 108s-1 using Tris-HCl as the scavenger, the ratio of single- to double-strand breaks for α particles is fivefold less than the corresponding ratios for γ irradiation. The repair of such radiation-induced single-strand breaks has been examined using a cell-free system derived from human whole-cell extracts. We show that the rejoining of single-strand breaks for both α-particle- and γ-irradiated plasmid is dependent upon the scavenging capacity and that the efficiency of rejoining of α-particle-induced single-strand breaks is significantly less than that observed for γ-ray-induced breaks. In addition, for DNA that had been irradiated under conditions that mimic the cellular environment with respect to the radical scavenging capacity, 50 of α-particle-induced single-strand breaks are converted to double-strand breaks, in contrast with only ∼12% conversion of γ-ray-induced single-strand breaks, indicating that the initial damage caused by α particles is more severe. These studies provide experimental evidence for increased clustering of damage which may have important implications for the induction of cancer by low-level α-particle sources such as domestic radon. 37 refs., 5 figs., 1 tab

  2. Efficient Capture and Isolation of Tumor-Related Circulating Cell-Free DNA from Cancer Patients Using Electroactive Conducting Polymer Nanowire Platforms

    Science.gov (United States)

    Jeon, SeungHyun; Lee, HyungJae; Bae, Kieun; Yoon, Kyong-Ah; Lee, Eun Sook; Cho, Youngnam

    2016-01-01

    Circulating cell-free DNA (cfDNA) is currently recognized as a key non-invasive biomarker for cancer diagnosis and progression and therapeutic efficacy monitoring. Because cfDNA has been detected in patients with diverse types of cancers, the use of efficient strategies to isolate cfDNA not only provides valuable insights into tumour biology, but also offers the potential for developing new cancer-specific targets. However, the challenges associated with conventional cfDNA extraction methods prevent their further clinical applications. Here, we developed a nanostructured conductive polymer platform for the efficient capture and release of circulating cfDNA and demonstrated its potential clinical utility using unprocessed plasma samples from patients with breast and lung cancers. Our results confirmed that the platform's enhanced efficiency allows tumor-specific circulating cfDNA to be recovered at high yield and purity. PMID:27162553

  3. Circulating Cell-Free DNA in Plasma of Locally Advanced Rectal Cancer Patients Undergoing Preoperative Chemoradiation: A Potential Diagnostic Tool for Therapy Monitoring

    Directory of Open Access Journals (Sweden)

    Matthias Zitt

    2008-01-01

    Full Text Available Circulating cell-free DNA opens up an interesting field for therapy monitoring, in particular during multimodal therapy protocols. The objective of this proof of principle study was to evaluate whether the amount of circulating plasma DNA has the potential to serve as a marker for therapy monitoring during the treatment course of locally advanced rectal cancer patients. We especially focused on kinetics of circulating DNA to assess whether variances in kinetics have the potential to discriminate between therapy responders and nonresponders.

  4. Serum cell-free DNA concentration in BALB/c mice with azoxymethane-dextran sodium sulfate-induced colorectal cancer

    OpenAIRE

    Virhan Novianry; Yulhasri Yulhasri; Kusmardi Kusmardi

    2015-01-01

    Background: Colorectal cancer is the third most common cancer in the United States with a mortality rate ranked second in 2012. Early diagnosis such as detection of DNA in serum or faeces at the polyp stage, will reduce colorectal cancer mortality. This study was conducted to analyze the concentration of cell-free DNA (cfDNA) as a tumor marker in colorectal carcinogenesis by using blood serum samples from BALB/c mice previously induced by azoxymethane (AOM) and promoted by dextran sodium sulf...

  5. Fetal Nucleic Acids in Maternal Circulation: A Genetic Resource for Noninvasive Prenatal Diagnosis

    OpenAIRE

    Banerjee, Monisha; Misra, Deepika

    2013-01-01

    Invasive prenatal diagnosis (PND) holds a multitude of psychological considerations for women, their partners, family and community as a whole. Earlier, the non-invasive screening methods for certain disorders were serum analytes or ultrasound with low sensitivity and high false positivity. The discovery of fetal DNA in maternal plasma has opened up an approach for noninvasive PND (NIPD). Presence of fetal cells and cell-free fetal DNA (cffDNA) in the blood of pregnant women has been accepted...

  6. Chimerism Analysis of Cell-Free DNA in Patients Treated with Hematopoietic Stem Cell Transplantation May Predict Early Relapse in Patients with Hematologic Malignancies

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    Mahmoud Aljurf

    2016-01-01

    Full Text Available Background. We studied DNA chimerism in cell-free DNA (cfDNA in patients treated with HSCT. Methods. Chimerism analysis was performed on CD3+ cells, polymorphonuclear (PMN cells, and cfDNA using 16 small tandem repeat loci. The resulting labeled PCR-products were size-fractionated and quantified. Results. Analyzing samples from 191 patients treated with HSCT for nonneoplastic hematologic disorders demonstrated that the cfDNA chimerism is comparable to that seen in PMN cells. Analyzing leukemia patients (N = 126 showed that, of 84 patients with 100% donor DNA in PMN, 16 (19% had evidence of clinical relapse and >10% recipient DNA in the plasma. Additional 16 patients of the 84 (19% showed >10% recipient DNA in plasma, but without evidence of relapse. Eight patients had mixed chimerism in granulocytes, lymphocytes, and plasma, but three of these patients had >10% recipient DNA in plasma compared to PMN cells and these three patients had clinical evidence of relapse. The remaining 34 patients showed 100% donor DNA in both PMN and lymphocytes, but cfDNA showed various levels of chimerism. Of these patients 14 (41% showed laboratory or clinical evidence of relapse and all had >10% recipient DNA in cfDNA. Conclusion. Monitoring patients after HSCT using cfDNA might be more reliable than cellular DNA in predicting early relapse.

  7. Chimerism Analysis of Cell-Free DNA in Patients Treated with Hematopoietic Stem Cell Transplantation May Predict Early Relapse in Patients with Hematologic Malignancies

    Science.gov (United States)

    Aljurf, Mahmoud; Abalkhail, Hala; Alseraihy, Amal; Mohamed, Said Y.; Ayas, Mouhab; Alsharif, Fahad; Alzahrani, Hazza; Al-Jefri, Abdullah; Aldawsari, Ghuzayel; Al-Ahmari, Ali; Belgaumi, Asim F.; Walter, Claudia Ulrike; El-Solh, Hassan; Rasheed, Walid; Albitar, Maher

    2016-01-01

    Background. We studied DNA chimerism in cell-free DNA (cfDNA) in patients treated with HSCT. Methods. Chimerism analysis was performed on CD3+ cells, polymorphonuclear (PMN) cells, and cfDNA using 16 small tandem repeat loci. The resulting labeled PCR-products were size-fractionated and quantified. Results. Analyzing samples from 191 patients treated with HSCT for nonneoplastic hematologic disorders demonstrated that the cfDNA chimerism is comparable to that seen in PMN cells. Analyzing leukemia patients (N = 126) showed that, of 84 patients with 100% donor DNA in PMN, 16 (19%) had evidence of clinical relapse and >10% recipient DNA in the plasma. Additional 16 patients of the 84 (19%) showed >10% recipient DNA in plasma, but without evidence of relapse. Eight patients had mixed chimerism in granulocytes, lymphocytes, and plasma, but three of these patients had >10% recipient DNA in plasma compared to PMN cells and these three patients had clinical evidence of relapse. The remaining 34 patients showed 100% donor DNA in both PMN and lymphocytes, but cfDNA showed various levels of chimerism. Of these patients 14 (41%) showed laboratory or clinical evidence of relapse and all had >10% recipient DNA in cfDNA. Conclusion. Monitoring patients after HSCT using cfDNA might be more reliable than cellular DNA in predicting early relapse. PMID:27006832

  8. Acceptance of non-invasive prenatal testing by cell free foetal DNA for foetal aneuploidy in a developing country: experience at a tertiary care centre in India

    Directory of Open Access Journals (Sweden)

    Namrata Kashyap

    2016-03-01

    Conclusions: Newer genomic technology involving cell free maternal DNA is a new storm in prenatal diagnosis. Its application in clinical practice is the need of the hour, however, the lack of awareness, high cost and unavailability of the test in the country appears to be a major limiting factor for its poor acceptability. [Int J Reprod Contracept Obstet Gynecol 2016; 5(3.000: 705-710

  9. Value of Quantitative and Qualitative Analyses of Circulating Cell-Free DNA as Diagnostic Tools for Hepatocellular Carcinoma

    Science.gov (United States)

    Liao, Wenjun; Mao, Yilei; Ge, Penglei; Yang, Huayu; Xu, Haifeng; Lu, Xin; Sang, Xinting; Zhong, Shouxian

    2015-01-01

    Abstract Qualitative and quantitative analyses of circulating cell-free DNA (cfDNA) are potential methods for the detection of hepatocellular carcinoma (HCC). Many studies have evaluated these approaches, but the results have been variable. This meta-analysis is the first to synthesize these published results and evaluate the use of circulating cfDNA values for HCC diagnosis. All articles that met our inclusion criteria were assessed using QUADAS guidelines after the literature research. We also investigated 3 subgroups in this meta-analysis: qualitative analysis of abnormal concentrations of circulating cfDNA; qualitative analysis of single-gene methylation alterations; and multiple analyses combined with alpha-fetoprotein (AFP). Statistical analyses were performed using the software Stata 12.0. We synthesized these published results and calculated accuracy measures (pooled sensitivity and specificity, positive/negative likelihood ratios [PLRs/NLRs], diagnostic odds ratios [DORs], and corresponding 95% confidence intervals [95% CIs]). Data were pooled using bivariate generalized linear mixed model. Furthermore, summary receiver operating characteristic curves and area under the curve (AUC) were used to summarize overall test performance. Heterogeneity and publication bias were also examined. A total of 2424 subjects included 1280 HCC patients in 22 studies were recruited in this meta-analysis. Pooled sensitivity and specificity, PLR, NLR, DOR, AUC, and CIs of quantitative analysis were 0.741 (95% CI: 0.610–0.840), 0.851 (95% CI: 0.718–0.927), 4.970 (95% CI: 2.694–9.169), 0.304 (95% CI: 0.205–0.451), 16.347 (95% CI: 8.250–32.388), and 0.86 (95% CI: 0.83–0.89), respectively. For qualitative analysis, the values were 0.538 (95% CI: 0.401–0.669), 0.944 (95% CI: 0.889–0.972), 9.545 (95% CI: 5.298–17.196), 0.490 (95% CI: 0.372–0.646), 19.491 (95% CI: 10.458–36.329), and 0.87 (95% CI: 0.84–0.90), respectively. After combining with AFP assay, the

  10. Detection of Clonal and Subclonal Copy-Number Variants in Cell-Free DNA from Patients with Breast Cancer Using a Massively Multiplexed PCR Methodology

    OpenAIRE

    Eser Kirkizlar; Bernhard Zimmermann; Tudor Constantin; Ryan Swenerton; Bin Hoang; Nicholas Wayham; Babiarz, Joshua E.; Zachary Demko; Robert J Pelham; Stephanie Kareht; Alexander L. Simon; Kristine N. Jinnett; Matthew Rabinowitz; Styrmir Sigurjonsson; Matthew Hill

    2015-01-01

    We demonstrate proof-of-concept for the use of massively multiplexed PCR and next-generation sequencing (mmPCR-NGS) to identify both clonal and subclonal copy-number variants (CNVs) in circulating tumor DNA. This is the first report of a targeted methodology for detection of CNVs in plasma. Using an in vitro model of cell-free DNA, we show that mmPCR-NGS can accurately detect CNVs with average allelic imbalances as low as 0.5%, an improvement over previously reported whole-genome sequencing a...

  11. Prospective blinded study of somatic mutation detection in cell-free DNA utilizing a targeted 54-gene next generation sequencing panel in metastatic solid tumor patients

    Science.gov (United States)

    Lanman, Richard B.; Mortimer, Stefanie; Zill, Oliver A.; Kim, Kyoung-Mee; Jang, Kee Taek; Kim, Seok-Hyung; Park, Se Hoon; Park, Joon Oh; Park, Young Suk; Lim, Ho Yeong; Eltoukhy, Helmy; Kang, Won Ki; Lee, Woo Yong; Kim, Hee-Cheol; Park, Keunchil; Lee, Jeeyun; Talasaz, AmirAli

    2015-01-01

    Sequencing of the mutant allele fraction of circulating cell-free DNA (cfDNA) derived from tumors is increasingly utilized to detect actionable genomic alterations in cancer. We conducted a prospective blinded study of a comprehensive cfDNA sequencing panel with 54 cancer genes. To evaluate the concordance between cfDNA and tumor DNA (tDNA), sequencing results were compared between cfDNA from plasma and genomic tumor DNA (tDNA). Utilizing next generation digital sequencing technology (DST), we profiled approximately 78,000 bases encoding 512 complete exons in the targeted genes in cfDNA from plasma. Seventy-five patients were prospectively enrolled between February 2013 and March 2014, including 61 metastatic cancer patients and 14 clinical stage II CRC patients with matched plasma and tissue samples. Using the 54-gene panel, we detected at least one somatic mutation in 44 of 61 tDNA (72.1%) and 29 of 44 (65.9%) cfDNA. The overall concordance rate of cfDNA to tDNA was 85.9%, when all detected mutations were considered. We collected serial cfDNAs during cetuximab-based treatment in 2 metastatic KRAS wild-type CRC patients, one with acquired resistance and one with primary resistance. We demonstrate newly emerged KRAS mutation in cfDNA 1.5 months before radiologic progression. Another patient had a newly emerged PIK3CA H1047R mutation on cfDNA analysis at progression during cetuximab/irinotecan chemotherapy with gradual increase in allele frequency from 0.8 to 2.1%. This blinded, prospective study of a cfDNA sequencing showed high concordance to tDNA suggesting that the DST approach may be used as a non-invasive biopsy-free alternative to conventional sequencing using tumor biopsy. PMID:26452027

  12. THE MATERNAL-FETAL MEDICINE: AN UPDATE

    OpenAIRE

    Vincenzo Berghella; Alessandro Ghidini; Giancarlo Mari; Mariarosaria Di Tommaso; Silvia Vannuccini; Filiberto Maria Severi; Felice Petraglia

    2013-01-01

    The development of Maternal-Fetal Medicine is contributing to an improvement of maternal well-being and of neonatal health, introducing a number of new and useful technologies. Advances in genomics in the field of prenatal screening and diagnosis allowed the discovery of fragments of cell-free fetal DNA in the maternal circulation and the use of chromosomal microarrays, which can test for microdeletions and microduplications in addition to aneuploidies. Color Doppler applications during pregn...

  13. Non-invasive detection of genomic imbalances in Hodgkin/Reed-Sternberg cells in early and advanced stage Hodgkin's lymphoma by sequencing of circulating cell-free DNA: a technical proof-of-principle study

    OpenAIRE

    Vandenberghe, Peter; Wlodarska, Iwona; Tousseyn, Thomas; Dehaspe, Luc; Dierickx, Daan; Verheecke, Magali; Uyttebroeck, Anne; Bechter, Oliver; Delforge, Michel; Vandecaveye, Vincent; Brison, Nathalie; Verhoef, Gregor; Legius, Eric; Amant, Frédéric; Vermeesch, Joris

    2015-01-01

    Hodgkin's lymphoma is one of the most common lymphoid neoplasms in young adults, but the low abundance of neoplastic Hodgkin/Reed-Sternberg cells in the tumour hampers the elucidation of its pathogenesis, biology, and diversity. After an incidental observation that genomic aberrations known to occur in Hodgkin's lymphoma were detectable in circulating cell-free DNA, this study was undertaken to investigate whether circulating cell-free DNA can be informative about genomic imbalances in Hodgki...

  14. THE MATERNAL-FETAL MEDICINE: AN UPDATE

    Directory of Open Access Journals (Sweden)

    Vincenzo Berghella

    2013-12-01

    Full Text Available The development of Maternal-Fetal Medicine is contributing to an improvement of maternal well-being and of neonatal health, introducing a number of new and useful technologies. Advances in genomics in the field of prenatal screening and diagnosis allowed the discovery of fragments of cell-free fetal DNA in the maternal circulation and the use of chromosomal microarrays, which can test for microdeletions and microduplications in addition to aneuploidies. Color Doppler applications during pregnancy are expanding exponentially and Doppler flow velocity waveforms indices have provided important information from maternal, placental and fetal circulation with clinical implications. Ultrasound monitoring of fetal growth represents a fundamental tool to evaluate fetal wellbeing and several methods have been developed to improve fetal weight estimation accuracy. The combination of new biophysical and biochemical markers is enriching Maternal-Fetal Medicine and more research will allow to improve pregnancy outcome.

  15. Analysis of 16S rRNA gene sequences and circulating cell-free DNA from plasma of chronic fatigue syndrome and non-fatigued subjects

    Directory of Open Access Journals (Sweden)

    Unger Elizabeth R

    2002-12-01

    Full Text Available Abstract Background The association of an infectious agent with chronic fatigue syndrome (CFS has been difficult and is further complicated by the lack of a known lesion or diseased tissue. Cell-free plasma DNA could serve as a sentinel of infection and disease occurring throughout the body. This type of systemic sample coupled with broad-range amplification of bacterial sequences was used to determine whether a bacterial pathogen was associated with CFS. Plasma DNA from 34 CFS and 55 non-fatigued subjects was assessed to determine plasma DNA concentration and the presence of bacterial 16S ribosomal DNA (rDNA sequences. Results DNA was isolated from 81 (91% of 89 plasma samples. The 55 non-fatigued subjects had higher plasma DNA concentrations than those with CFS (average 151 versus 91 ng and more CFS subjects (6/34, 18% had no detectable plasma DNA than non-fatigued subjects (2/55, 4%, but these differences were not significant. Bacterial sequences were detected in 23 (26% of 89. Only 4 (14% CFS subjects had 16S rDNA sequences amplified from plasma compared with 17 (32% of the non-fatigued (P = 0.03. All but 1 of the 23 16S rDNA amplicon-positive subjects had five or more unique sequences present. Conclusions CFS subjects had slightly lower concentrations or no detectable plasma DNA than non-fatigued subjects. There was a diverse array of 16S rDNA sequences in plasma DNA from both CFS and non-fatigued subjects. There were no unique, previously uncharacterized or predominant 16S rDNA sequences in either CFS or non-fatigued subjects.

  16. Bleomycin-induced DNA synthesis in a cell-free system using a permeable mouse sarcoma cell Extract.

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    Seki,Shuji

    1987-10-01

    Full Text Available To investigate factors involved in excision repair DNA synthesis, a soluble extract was prepared from permeable mouse sarcoma (SR-C3H/He cells by homogenization and ultracentrifugation. DNA synthesis measured by using native calf thymus DNA as the template-primer and the extract as the polymerase source showed low activity. The DNA synthesis was enhanced more than ten-fold by the addition of an appropriate concentration of bleomycin, a radiomimetic DNA-damaging drug. Using selective inhibitors of DNA polymerases, it was shown that the DNA polymerase involved in the bleomycin-induced DNA synthesis was DNA polymerase beta. In addition to DNA polymerase beta, an exonuclease which converts bleomycin-damaged DNA into suitable template-primers for repair DNA synthesis appeared to be present in the permeable cell extract.

  17. A novel strategy for highly efficient isolation and analysis of circulating tumor-specific cell-free DNA from lung cancer patients using a reusable conducting polymer nanostructure.

    Science.gov (United States)

    Lee, HyungJae; Jeon, SeungHyun; Seo, Jin-Suck; Goh, Sung-Ho; Han, Ji-Youn; Cho, Youngnam

    2016-09-01

    We have developed a reusable nanostructured polypyrrole nanochip and demonstrated its use in the electric field-mediated recovery of circulating cell-free DNA (cfDNA) from the plasma of lung cancer patients. Although cfDNA has been recognized and widely studied as a versatile and promising biomarker for the diagnosis and prognosis of cancers, the lack of efficient strategies to directly isolate cfDNA from the plasma has become a great hindrance to its potential clinical use. As a proof-of-concept study, we demonstrated a technique for the rapid and efficient isolation of cfDNA with high yield and purity. In particular, the synergistic effects of the electro-activity and the nanostructured features of the polypyrrole polymer enabled repeated retrieval of cfDNA using a single platform. Moreover, polypyrrole nanochip facilitated the amplification of tumor-specific DNA fragments from the plasma samples of patients with lung cancer characterized by mutations in exons 21 of the epidermal growth factor receptor gene (EGFR). Overall, the proposed polypyrrole nanochip has enormous potential for industrial and clinical applications with significantly enhanced efficiency in the recovery of tumor-associated circulating cfDNA. This may ultimately contribute to more robust and reliable evaluation of gene mutations in peripheral blood. PMID:27294542

  18. Analytical and Clinical Validation of a Digital Sequencing Panel for Quantitative, Highly Accurate Evaluation of Cell-Free Circulating Tumor DNA

    Science.gov (United States)

    Zill, Oliver A.; Sebisanovic, Dragan; Lopez, Rene; Blau, Sibel; Collisson, Eric A.; Divers, Stephen G.; Hoon, Dave S. B.; Kopetz, E. Scott; Lee, Jeeyun; Nikolinakos, Petros G.; Baca, Arthur M.; Kermani, Bahram G.; Eltoukhy, Helmy; Talasaz, AmirAli

    2015-01-01

    Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital SequencingTM is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient’s cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing. PMID:26474073

  19. Analytical and Clinical Validation of a Digital Sequencing Panel for Quantitative, Highly Accurate Evaluation of Cell-Free Circulating Tumor DNA.

    Directory of Open Access Journals (Sweden)

    Richard B Lanman

    Full Text Available Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital Sequencing™ is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999% enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient's cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing.

  20. Cell-free fetal DNA in the maternal circulation originates from the cytotrophoblast : proof from an unique case

    NARCIS (Netherlands)

    Hochstenbach, Ron; Nikkels, Peter G J; Elferink, Martin G; Oudijk, Martijn A; van Oppen, Carla; van Zon, Patrick; van Harssel, Jeske; Schuring-Blom, Heleen; Page-Christiaens, Godelieve C M L

    2015-01-01

    Noninvasive prenatal testing (NIPT) and direct karyotyping of cytotrophoblast were normal for a male fetus, but cultured chorionic villus mesenchymal cells and umbilical cord fibroblasts showed nonmosaic trisomy 18. This observation provides direct evidence for the cytotrophoblastic origin of cell-f

  1. Pleural fluid cell-free DNA integrity index to identify cytologically negative malignant pleural effusions including mesotheliomas

    International Nuclear Information System (INIS)

    The diagnosis of malignant pleural effusions (MPE) is often clinically challenging, especially if the cytology is negative for malignancy. DNA integrity index has been reported to be a marker of malignancy. The aim of this study was to evaluate the utility of pleural fluid DNA integrity index in the diagnosis of MPE. We studied 75 pleural fluid and matched serum samples from consecutive subjects. Pleural fluid and serum ALU DNA repeats [115bp, 247bp and 247bp/115bp ratio (DNA integrity index)] were assessed by real-time quantitative PCR. Pleural fluid and serum mesothelin levels were quantified using ELISA. Based on clinico-pathological evaluation, 52 subjects had MPE (including 16 mesotheliomas) and 23 had benign effusions. Pleural fluid DNA integrity index was higher in MPE compared with benign effusions (1.2 vs. 0.8; p<0.001). Cytology had a sensitivity of 55% in diagnosing MPE. If cytology and pleural fluid DNA integrity index were considered together, they exhibited 81% sensitivity and 87% specificity in distinguishing benign and malignant effusions. In cytology-negative pleural effusions (35 MPE and 28 benign effusions), elevated pleural fluid DNA integrity index had an 81% positive predictive value in detecting MPEs. In the detection of mesothelioma, at a specificity of 90%, pleural fluid DNA integrity index had similar sensitivity to pleural fluid and serum mesothelin (75% each respectively). Pleural fluid DNA integrity index is a promising diagnostic biomarker for identification of MPEs, including mesothelioma. This biomarker may be particularly useful in cases of MPE where pleural aspirate cytology is negative, and could guide the decision to undertake more invasive definitive testing. A prospective validation study is being undertaken to validate our findings and test the clinical utility of this biomarker for altering clinical practice

  2. Identification of Human N-Myristoylated Proteins from Human Complementary DNA Resources by Cell-Free and Cellular Metabolic Labeling Analyses

    Science.gov (United States)

    Takamitsu, Emi; Otsuka, Motoaki; Haebara, Tatsuki; Yano, Manami; Matsuzaki, Kanako; Kobuchi, Hirotsugu; Moriya, Koko; Utsumi, Toshihiko

    2015-01-01

    To identify physiologically important human N-myristoylated proteins, 90 cDNA clones predicted to encode human N-myristoylated proteins were selected from a human cDNA resource (4,369 Kazusa ORFeome project human cDNA clones) by two bioinformatic N-myristoylation prediction systems, NMT-The MYR Predictor and Myristoylator. After database searches to exclude known human N-myristoylated proteins, 37 cDNA clones were selected as potential human N-myristoylated proteins. The susceptibility of these cDNA clones to protein N-myristoylation was first evaluated using fusion proteins in which the N-terminal ten amino acid residues were fused to an epitope-tagged model protein. Then, protein N-myristoylation of the gene products of full-length cDNAs was evaluated by metabolic labeling experiments both in an insect cell-free protein synthesis system and in transfected human cells. As a result, the products of 13 cDNA clones (FBXL7, PPM1B, SAMM50, PLEKHN, AIFM3, C22orf42, STK32A, FAM131C, DRICH1, MCC1, HID1, P2RX5, STK32B) were found to be human N-myristoylated proteins. Analysis of the role of protein N-myristoylation on the intracellular localization of SAMM50, a mitochondrial outer membrane protein, revealed that protein N-myristoylation was required for proper targeting of SAMM50 to mitochondria. Thus, the strategy used in this study is useful for the identification of physiologically important human N-myristoylated proteins from human cDNA resources. PMID:26308446

  3. Identification of Human N-Myristoylated Proteins from Human Complementary DNA Resources by Cell-Free and Cellular Metabolic Labeling Analyses.

    Directory of Open Access Journals (Sweden)

    Emi Takamitsu

    Full Text Available To identify physiologically important human N-myristoylated proteins, 90 cDNA clones predicted to encode human N-myristoylated proteins were selected from a human cDNA resource (4,369 Kazusa ORFeome project human cDNA clones by two bioinformatic N-myristoylation prediction systems, NMT-The MYR Predictor and Myristoylator. After database searches to exclude known human N-myristoylated proteins, 37 cDNA clones were selected as potential human N-myristoylated proteins. The susceptibility of these cDNA clones to protein N-myristoylation was first evaluated using fusion proteins in which the N-terminal ten amino acid residues were fused to an epitope-tagged model protein. Then, protein N-myristoylation of the gene products of full-length cDNAs was evaluated by metabolic labeling experiments both in an insect cell-free protein synthesis system and in transfected human cells. As a result, the products of 13 cDNA clones (FBXL7, PPM1B, SAMM50, PLEKHN, AIFM3, C22orf42, STK32A, FAM131C, DRICH1, MCC1, HID1, P2RX5, STK32B were found to be human N-myristoylated proteins. Analysis of the role of protein N-myristoylation on the intracellular localization of SAMM50, a mitochondrial outer membrane protein, revealed that protein N-myristoylation was required for proper targeting of SAMM50 to mitochondria. Thus, the strategy used in this study is useful for the identification of physiologically important human N-myristoylated proteins from human cDNA resources.

  4. BRAF Mutation Testing in Cell-Free DNA from the Plasma of Patients with Advanced Cancers Using a Rapid, Automated Molecular Diagnostics System.

    Science.gov (United States)

    Janku, Filip; Huang, Helen J; Claes, Bart; Falchook, Gerald S; Fu, Siqing; Hong, David; Ramzanali, Nishma M; Nitti, Giovanni; Cabrilo, Goran; Tsimberidou, Apostolia M; Naing, Aung; Piha-Paul, Sarina A; Wheler, Jennifer J; Karp, Daniel D; Holley, Veronica R; Zinner, Ralph G; Subbiah, Vivek; Luthra, Rajyalakshmi; Kopetz, Scott; Overman, Michael J; Kee, Bryan K; Patel, Sapna; Devogelaere, Benoit; Sablon, Erwin; Maertens, Geert; Mills, Gordon B; Kurzrock, Razelle; Meric-Bernstam, Funda

    2016-06-01

    Cell-free (cf) DNA from plasma offers an easily obtainable material for BRAF mutation analysis for diagnostics and response monitoring. In this study, plasma-derived cfDNA samples from patients with progressing advanced cancers or malignant histiocytosis with known BRAF(V600) status from formalin-fixed paraffin-embedded (FFPE) tumors were tested using a prototype version of the Idylla BRAF Mutation Test, a fully integrated real-time PCR-based test with turnaround time about 90 minutes. Of 160 patients, BRAF(V600) mutations were detected in 62 (39%) archival FFPE tumor samples and 47 (29%) plasma cfDNA samples. The two methods had overall agreement in 141 patients [88%; κ, 0.74; SE, 0.06; 95% confidence interval (CI), 0.63-0.85]. Idylla had a sensitivity of 73% (95% CI, 0.60-0.83) and specificity of 98% (95% CI, 0.93-1.00). A higher percentage, but not concentration, of BRAF(V600) cfDNA in the wild-type background (>2% vs. ≤ 2%) was associated with shorter overall survival (OS; P = 0.005) and in patients with BRAF mutations in the tissue, who were receiving BRAF/MEK inhibitors, shorter time to treatment failure (TTF; P = 0.001). Longitudinal monitoring demonstrated that decreasing levels of BRAF(V600) cfDNA were associated with longer TTF (P = 0.045). In conclusion, testing for BRAF(V600) mutations in plasma cfDNA using the Idylla BRAF Mutation Test has acceptable concordance with standard testing of tumor tissue. A higher percentage of mutant BRAF(V600) in cfDNA corresponded with shorter OS and in patients receiving BRAF/MEK inhibitors also with shorter TTF. Mol Cancer Ther; 15(6); 1397-404. ©2016 AACR. PMID:27207774

  5. Divalent cation ionophores stimulate resorption and inhibit DNA synthesis in cultured fetal rat bone

    Energy Technology Data Exchange (ETDEWEB)

    Lorenzo, J.A.; Raisz, L.G.

    1981-06-01

    Two divalent cation ionophores, A23187 and Ionomycin, which are selective for calcium, stimulated the resorption of fetal rat long bones in organ culture at 0.1 to 1 micromolar but not at higher concentrations. Both agents inhibited DNA synthesis at concentrations that stimulated resorption. These results might explain the differences in ionophore effects on bone previously reported, and they imply that cell replication is not required for osteoclast formation in fetal rat long bone cultures.

  6. Detection of Clonal and Subclonal Copy-Number Variants in Cell-Free DNA from Patients with Breast Cancer Using a Massively Multiplexed PCR Methodology

    Directory of Open Access Journals (Sweden)

    Eser Kirkizlar

    2015-10-01

    Using an in vitro model of cell-free DNA, we show that mmPCR-NGS can accurately detect CNVs with average allelic imbalances as low as 0.5%, an improvement over previously reported whole-genome sequencing approaches. Our method revealed differences in the spectrum of CNVs detected in tumor tissue subsections and matching plasma samples from 11 patients with stage II breast cancer. Moreover, we showed that liquid biopsies are able to detect subclonal mutations that may be missed in tumor tissue biopsies. We anticipate that this mmPCR-NGS methodology will have broad applicability for the characterization, diagnosis, and therapeutic monitoring of CNV-enriched cancers, such as breast, ovarian, and lung cancer.

  7. Pleural fluid cell-free DNA integrity index to identify cytologically negative malignant pleural effusions including mesotheliomas

    Directory of Open Access Journals (Sweden)

    Sriram Krishna B

    2012-09-01

    Full Text Available Abstract Background The diagnosis of malignant pleural effusions (MPE is often clinically challenging, especially if the cytology is negative for malignancy. DNA integrity index has been reported to be a marker of malignancy. The aim of this study was to evaluate the utility of pleural fluid DNA integrity index in the diagnosis of MPE. Methods We studied 75 pleural fluid and matched serum samples from consecutive subjects. Pleural fluid and serum ALU DNA repeats [115bp, 247bp and 247bp/115bp ratio (DNA integrity index] were assessed by real-time quantitative PCR. Pleural fluid and serum mesothelin levels were quantified using ELISA. Results Based on clinico-pathological evaluation, 52 subjects had MPE (including 16 mesotheliomas and 23 had benign effusions. Pleural fluid DNA integrity index was higher in MPE compared with benign effusions (1.2 vs. 0.8; p Conclusion Pleural fluid DNA integrity index is a promising diagnostic biomarker for identification of MPEs, including mesothelioma. This biomarker may be particularly useful in cases of MPE where pleural aspirate cytology is negative, and could guide the decision to undertake more invasive definitive testing. A prospective validation study is being undertaken to validate our findings and test the clinical utility of this biomarker for altering clinical practice.

  8. Whole-genome bisulfite sequencing of cell-free DNA identifies signature associated with metastatic breast cancer

    OpenAIRE

    Legendre, Christophe; Gooden, Gerald C.; Johnson, Kyle; Martinez, Rae Anne; Liang, Winnie S.; Salhia, Bodour

    2015-01-01

    Background A number of clinico-pathological criteria and molecular profiles have been used to stratify patients into high- and low-risk groups. Currently, there are still no effective methods to determine which patients harbor micrometastatic disease after standard breast cancer therapy and who will eventually develop local or distant recurrence. The purpose of our study was to identify circulating DNA methylation changes that can be used for prediction of metastatic breast cancer (MBC). Resu...

  9. Detection of methylated septin 9 in tissue and plasma of colorectal patients with neoplasia and the relationship to the amount of circulating cell-free DNA.

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    Kinga Tóth

    Full Text Available Determination of methylated Septin 9 (mSEPT9 in plasma has been shown to be a sensitive and specific biomarker for colorectal cancer (CRC. However, the relationship between methylated DNA in plasma and colon tissue of the same subjects has not been reported.Plasma and matching biopsy samples were collected from 24 patients with no evidence of disease (NED, 26 patients with adenoma and 34 patients with CRC. Following bisulfite conversion of DNA a commercial RT-PCR assay was used to determine the total amount of DNA in each sample and the fraction of mSEPT9 DNA. The Septin-9 protein was assessed using immunohistochemistry.The percent of methylated reference (PMR values for SEPT9 above a PMR threshold of 1% were detected in 4.2% (1/24 of NED, 100% (26/26 of adenoma and 97.1% (33/34 of CRC tissues. PMR differences between NED vs. adenoma and NED vs. CRC comparisons were significant (p<0.001. In matching plasma samples using a PMR cut-off level of 0.01%, SEPT9 methylation was 8.3% (2/24 of NED, 30.8% (8/26 of adenoma and 88.2% (30/34 of CRC. Significant PMR differences were observed between NED vs. CRC (p<0.01 and adenoma vs. CRC (p<0.01. Significant differences (p<0.01 were found in the amount of cfDNA (circulating cell-free DNA between NED and CRC, and a modest correlation was observed between mSEPT9 concentration and cfDNA of cancer (R2 = 0.48. The level of Septin-9 protein in tissues was inversely correlated to mSEPT9 levels with abundant expression in normals, and diminished expression in adenomas and tumors.Methylated SEPT9 was detected in all tissue samples. In plasma samples, elevated mSEPT9 values were detected in CRC, but not in adenomas. Tissue levels of mSEPT9 alone are not sufficient to predict mSEPT9 levels in plasma. Additional parameters including the amount of cfDNA in plasma appear to also play a role.

  10. High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients.

    Science.gov (United States)

    Kukita, Yoji; Matoba, Ryo; Uchida, Junji; Hamakawa, Takuya; Doki, Yuichiro; Imamura, Fumio; Kato, Kikuya

    2015-08-01

    Circulating tumour DNA (ctDNA) is an emerging field of cancer research. However, current ctDNA analysis is usually restricted to one or a few mutation sites due to technical limitations. In the case of massively parallel DNA sequencers, the number of false positives caused by a high read error rate is a major problem. In addition, the final sequence reads do not represent the original DNA population due to the global amplification step during the template preparation. We established a high-fidelity target sequencing system of individual molecules identified in plasma cell-free DNA using barcode sequences; this system consists of the following two steps. (i) A novel target sequencing method that adds barcode sequences by adaptor ligation. This method uses linear amplification to eliminate the errors introduced during the early cycles of polymerase chain reaction. (ii) The monitoring and removal of erroneous barcode tags. This process involves the identification of individual molecules that have been sequenced and for which the number of mutations have been absolute quantitated. Using plasma cell-free DNA from patients with gastric or lung cancer, we demonstrated that the system achieved near complete elimination of false positives and enabled de novo detection and absolute quantitation of mutations in plasma cell-free DNA. PMID:26126624

  11. High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients

    Science.gov (United States)

    Kukita, Yoji; Matoba, Ryo; Uchida, Junji; Hamakawa, Takuya; Doki, Yuichiro; Imamura, Fumio; Kato, Kikuya

    2015-01-01

    Circulating tumour DNA (ctDNA) is an emerging field of cancer research. However, current ctDNA analysis is usually restricted to one or a few mutation sites due to technical limitations. In the case of massively parallel DNA sequencers, the number of false positives caused by a high read error rate is a major problem. In addition, the final sequence reads do not represent the original DNA population due to the global amplification step during the template preparation. We established a high-fidelity target sequencing system of individual molecules identified in plasma cell-free DNA using barcode sequences; this system consists of the following two steps. (i) A novel target sequencing method that adds barcode sequences by adaptor ligation. This method uses linear amplification to eliminate the errors introduced during the early cycles of polymerase chain reaction. (ii) The monitoring and removal of erroneous barcode tags. This process involves the identification of individual molecules that have been sequenced and for which the number of mutations have been absolute quantitated. Using plasma cell-free DNA from patients with gastric or lung cancer, we demonstrated that the system achieved near complete elimination of false positives and enabled de novo detection and absolute quantitation of mutations in plasma cell-free DNA. PMID:26126624

  12. Detection of Clonal and Subclonal Copy-Number Variants in Cell-Free DNA from Patients with Breast Cancer Using a Massively Multiplexed PCR Methodology

    Science.gov (United States)

    Kirkizlar, Eser; Zimmermann, Bernhard; Constantin, Tudor; Swenerton, Ryan; Hoang, Bin; Wayham, Nicholas; Babiarz, Joshua E.; Demko, Zachary; Pelham, Robert J.; Kareht, Stephanie; Simon, Alexander L.; Jinnett, Kristine N.; Rabinowitz, Matthew; Sigurjonsson, Styrmir; Hill, Matthew

    2015-01-01

    We demonstrate proof-of-concept for the use of massively multiplexed PCR and next-generation sequencing (mmPCR-NGS) to identify both clonal and subclonal copy-number variants (CNVs) in circulating tumor DNA. This is the first report of a targeted methodology for detection of CNVs in plasma. Using an in vitro model of cell-free DNA, we show that mmPCR-NGS can accurately detect CNVs with average allelic imbalances as low as 0.5%, an improvement over previously reported whole-genome sequencing approaches. Our method revealed differences in the spectrum of CNVs detected in tumor tissue subsections and matching plasma samples from 11 patients with stage II breast cancer. Moreover, we showed that liquid biopsies are able to detect subclonal mutations that may be missed in tumor tissue biopsies. We anticipate that this mmPCR-NGS methodology will have broad applicability for the characterization, diagnosis, and therapeutic monitoring of CNV-enriched cancers, such as breast, ovarian, and lung cancer. PMID:26500031

  13. DNA methylation in the human placenta and fetal growth (Review)

    OpenAIRE

    KOUKOURA, OURANIA; Sifakis, Stavros; Spandidos, Demetrios A.

    2012-01-01

    Throughout in utero development, the placenta plays a key role in controlling growth and development. The placenta acts not only as a gatekeeper of nutrient and waste exchange between mother and developing fetus, but also as a regulator of the intrauterine environment. Its functions can be influenced by the environment encountered throughout pregnancy, thereby altering the appropriate genetic programming needed to allow for appropriate fetal growth. Epigenetic alterations related to environme...

  14. Serum cell-free DNA concentration in BALB/c mice with azoxymethane-dextran sodium sulfate-induced colorectal cancer

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    Virhan Novianry

    2015-04-01

    Full Text Available Background: Colorectal cancer is the third most common cancer in the United States with a mortality rate ranked second in 2012. Early diagnosis such as detection of DNA in serum or faeces at the polyp stage, will reduce colorectal cancer mortality. This study was conducted to analyze the concentration of cell-free DNA (cfDNA as a tumor marker in colorectal carcinogenesis by using blood serum samples from BALB/c mice previously induced by azoxymethane (AOM and promoted by dextran sodium sulfate (DSS.Methods: This experimental animal study used 6 BALB/c mice which had serial intervention in a certain time frame. The first serum samples were taken before induction of carcinogenesis (week-0; then AOM induction of carcinogenesis followed and the second sampling one week after AOM intervention (week-1. Subsequently, promotion of carcinogenesis followed with DSS and the third sampling one week after this intervention (week-2. The fourth sampling was 5 weeks after AOM-DSS intervention (week-6. Quantification of the serum cfDNA was performed with SYBR-Green II fluorescence using Rotor Gene 6000 as a reference. Histopathological examination verified induction of carcinogenesis. For statistical analysis paired T-test was used.Results: Concentration of serum cfDNA showed significant difference between sampling group at week-0 (1238.49 ± 674.84 pg/µL and sampling group at week-6 (2244.04 ± 726.57 pg/µL the latter group showing pre-cancerous histopathology. Slightly increased cfDNA at week-1 with AOM induction (1358.57 ± 803.81 pg/µL and week-2 after DSS promotion (1317.23 ± 735.92 pg/µL were not significantly different from week-0 samples.Conclusion: The concentration of cfDNA in the serum of BALB/c mice 5 weeks after AOM induction of carcinogenesis and DSS promotion is significantly higher than before induction.

  15. Evaluation of KRAS Gene Expression and LCS6 Variant in Genomic and Cell-Free DNA of Iranian Women With Endometriosis

    Science.gov (United States)

    Farahani, Maryam Shahrabi; Moghaddam, Soheila Amini; Mahdian, Reza

    2015-01-01

    Since the activation of KRAS results in de novo endometriosis in mice, KRAS is regarded as a crucial gene in ectopic endometrial implantation. Recently, it has been reported that 31% of women with endometriosis have KRAS let-7 complementary binding site 6 single-nucleotide polymorphism (LCS6 SNP). This study addresses the correlation between KRAS LCS6 SNP and endometriosis in a case–control study. To detect probable somatic mutation in ectopic endometrial tissue, we evaluated LCS6 SNP in cell-free DNA samples. Quantitative real-time reverse transcription-polymerase chain reaction was performed to determine the expression of KRAS transcripts in eutopic endometrial tissue. Our results suggest that the variant is not associated with the development of endometriosis in Iranian women. We observed higher levels of KRAS messenger RNA (mRNA) expression in eutopic endometrium of patients with endometriosis compared to controls. Although, the KRAS LCS6 is neither constitutional nor somatic biomarker for endometriosis, increased expression ratio of KRAS mRNA indicates its role in the implantation of endometrial tissue outside the uterine cavity. PMID:25361550

  16. Quantitative assessment of BRAF V600 mutant circulating cell-free tumor DNA as a tool for therapeutic monitoring in metastatic melanoma patients treated with BRAF/MEK inhibitors

    OpenAIRE

    SCHREUER, MAX; Meersseman, Geert; Van Den Herrewegen, Sari; Jansen, Yanina; Chevolet, Ines; Bott, Ambre; Wilgenhof, Sofie; Seremet, Teofila; Jacobs, Bart; Buyl, Ronald; Maertens, Geert; Neyns, Bart

    2016-01-01

    Background BRAF V600 mutant circulating cell-free tumor DNA (BRAF V600mut ctDNA) could serve as a specific biomarker in patients with BRAF V600 mutant melanoma. We analyzed the value of BRAF V600mut ctDNA from plasma as a monitoring tool for advanced melanoma patients treated with BRAF/MEK inhibitors. Methods Allele-specific quantitative PCR analysis for BRAF V600 E/E2/D/K/R/M mutations was performed on DNA extracted from plasma of patients with known BRAF V600 mutant melanoma who were treate...

  17. Quantification and dynamic monitoring of EGFR T790M in plasma cell-free DNA by digital PCR for prognosis of EGFR-TKI treatment in advanced NSCLC.

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    Zhijie Wang

    Full Text Available BACKGROUND: Among advanced non-small cell lung cancer (NSCLC patients with an acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI, about 50% carry the T790M mutation, but this frequency in EGFR-TKI-naïve patients and dynamic change during therapy remains unclear. This study investigated the quantification and dynamic change of T790M mutation in plasma cell-free DNA (cf-DNA of advanced NSCLC patients to assess the clinical outcomes of EGFR-TKI therapy. MATERIALS AND METHODS: We retrospectively investigated 135 patients with advanced NSCLC who obtained progression-free survival (PFS after EGFR-TKI for >6 months for their EGFR sensitive mutations and T790M mutation in matched pre- and post-TKI plasma samples, using denaturing high-performance liquid chromatography (DHPLC, amplification refractory mutation system (ARMS, and digital-PCR (D-PCR. Real-time PCR was performed to measure c-MET amplification. RESULTS: Detection limit of D-PCR in assessing the T790M mutation was approximately 0.03%. D-PCR identified higher frequency of T790M than ARMS in pre-TKI (31.3% vs. 5.5% and post-TKI (43.0% vs. 25.2% plasma samples. Patients with pre-TKI T790M showed inferior PFS (8.9 vs. 12.1 months, p = 0.007 and overall survival (OS, 19.3 vs. 31.9 months, p = 0.001 compared with those without T790M. In patients harboring EGFR sensitive mutation, high quantities of pre-TKI T790M predicted poorer PFS (p = 0.001 on EGFR-TKI than low ones. Moreover, patients who experienced increased quantity of T790M during EGFR-TKI treatment showed superior PFS and OS compared with those with decreased changes (p = 0.044 and p = 0.015, respectively. CONCLUSION: Qualitative and quantitative T790M in plasma cf-DNA by D-PCR provided a non-invasive and sensitive assay to predict EGFR-TKI prognosis.

  18. Motivations for undertaking DNA sequencing-based non-invasive prenatal testing for fetal aneuploidy: a qualitative study with early adopter patients in Hong Kong.

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    Huso Yi

    Full Text Available BACKGROUND: A newly introduced cell-free fetal DNA sequencing based non-invasive prenatal testing (DNA-NIPT detects Down syndrome with sensitivity of 99% at early gestational stage without risk of miscarriage. Attention has been given to its public health implications; little is known from consumer perspectives. This qualitative study aimed to explore women's motivations for using, and perceptions of, DNA-NIPT in Hong Kong. METHODS AND FINDINGS: In-depth interviews were conducted with 45 women who had undertaken DNA-NIPT recruited by purposive sampling based on socio-demographic and clinical characteristics. The sample included 31 women identified as high-risk from serum and ultrasound based Down syndrome screening (SU-DSS. Thematic narrative analysis examined informed-decision making of the test and identified the benefits and needs. Women outlined a number of reasons for accessing DNA-NIPT: reducing the uncertainty associated with risk probability-based results from SU-DSS, undertaking DNA-NIPT as a comprehensive measure to counteract risk from childbearing especially at advanced age, perceived predictive accuracy and absence of risk of harm to fetus. Accounts of women deemed high-risk or not high-risk are distinctive in a number of respects. High-risk women accessed DNA-NIPT to get a clearer idea of their risk. This group perceived SU-DSS as an unnecessary and confusing procedure because of its varying, protocol-dependent detection rates. Those women not deemed high-risk, in contrast, undertook DNA-NIPT for psychological assurance and to reduce anxiety even after receiving the negative result from SU-DSS. CONCLUSIONS: DNA-NIPT was regarded positively by women who chose this method of screening over the routine, less expensive testing options. Given its perceived utility, health providers need to consider whether DNA-NIPT should be offered as part of universal routine care to women at high-risk for fetal aneuploidy. If this is the case, then

  19. Noninvasive Antenatal Determination of Fetal Blood Group Using Next-Generation Sequencing

    DEFF Research Database (Denmark)

    Rieneck, Klaus; Clausen, Frederik Banch; Dziegiel, Morten Hanefeld

    2015-01-01

    Hemolytic disease of the fetus and newborn (HDFN) is a condition characterized by a decreased lifespan of fetal red blood cells caused by maternally produced allospecific antibodies transferred to the fetus during pregnancy. The antibodies bind to the corresponding blood group antigens on fetal red...... blood cells and induce hemolysis. Cell-free DNA derived from the conceptus circulates in maternal blood. Using next-generation sequencing (NGS), it can be determined if this cell-free fetal DNA encodes the corresponding blood group antigen that is the target of the maternal allospecific antibodies....... This determination carries no risk to the fetus. It is important to determine if the fetus is at risk of hemolysis to enable timely intervention. Many tests for blood groups are based solely on the presence or absence of a single nucleotide polymorphism (SNP). Antenatal determination of fetal blood group by NGS...

  20. Integration of noninvasive prenatal prediction of fetal blood group into clinical prenatal care

    DEFF Research Database (Denmark)

    Clausen, Frederik Banch

    2014-01-01

    Incompatibility of red blood cell blood group antigens between a pregnant woman and her fetus can cause maternal immunization and, consequently, hemolytic disease of the fetus and newborn. Noninvasive prenatal testing of cell-free fetal DNA can be used to assess the risk of hemolytic disease...

  1. Application of Fetal DNA in Maternal Plasma in Noninvasive Prenatal Diagnosis

    Institute of Scientific and Technical Information of China (English)

    赵茵; 邹丽

    2004-01-01

    Summary: To explore the application of fetal DNA in maternal plasma for noninvasive prenatal diagnosis, the DNA template was extracted by hydroxybenzene-chloroform from 44 maternal (7-41weeks) plasma. The Fetus-derived Y sequence DYZ-1 gene (149bp) was chosen to be amplified by PCR. The fragment was identified in all the plasma of male bearing pregnant women with the diagnostic accordance rate being 100.00 %. Two of the 22 female bearing pregnant women had false positive results. Among the 44 pregnant women, the diagnostic accordance rate was 88. 89 % at early pregnant stage, 100.00 % at medium pregnant stage, and 96.55 % at late stage respectively.The final accuracy of 95. 45 % was obtained in all cases. It was concluded that by means of hydroxybenzene-chloroform extraction the authors of this article promoted the concentration and purity of the DNA template, and diagnosed more accurately. The results showed that free fetal DNA in the maternal plasma could be regarded as the gene resource for noninvasive prenatal diagnosis.

  2. Non-invasive prenatal diagnosis for fetal sex determination: benefits and disadvantages from the service users' perspective

    OpenAIRE

    Lewis, Celine; Hill, Melissa; Skirton, Heather; Chitty, Lyn S.

    2012-01-01

    Prenatal fetal sex determination is clinically indicated for women who are at risk of having a child with a serious genetic disorder affecting a particular sex. Ultrasound has been the traditional method used, but early fetal sex determination using non-invasive prenatal diagnosis (NIPD) can now be performed using cell-free fetal DNA in maternal plasma. The study aim was to assess the views and experiences of service users who had used NIPD for fetal sex determination. In this paper, we repor...

  3. The diagnostic potential of maternal plasma in detecting fetal diseases by DNA test

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    Saha Biswajit

    2004-01-01

    Full Text Available Conventionally, DNA based investigations for fetal diseases are done by chorionic villous sampling and amniocentesis. Both are invasive techniques. Recently, molecular diagnosis has also been made possible in early pregnancy from maternal blood which is noninvasive and advantageous. Most of the researches have tried to identify the Y chromosome marker(s to detect a male fetus and paternally inherited allele. This is currently helpful to detect a very few genetic disorders including Rh D status in Rh negative women in early pregnancy and preeclampsia a few weeks preceding the clinical onset. This is a potential area for prenatal diagnosis in future.

  4. Detection of fetal mutations causing hemoglobinopathies by non-invasive prenatal diagnosis from maternal plasma

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    E D′Souza

    2013-01-01

    Full Text Available Background: Prenatal diagnosis of hemoglobinopathies enables couples at risk to have a healthy child. Currently used fetal sampling procedures are invasive with some risk of miscarriage. A non-invasive approach to obtain fetal deoxyribonucleic acid (DNA for diagnosis would eliminate this risk. Aim: To develop and evaluate a non-invasive prenatal diagnostic approach for hemoglobinopathies using cell-free fetal DNA circulating in the maternal plasma. Settings and Design: Couples referred to us for prenatal diagnosis of hemoglobinopathies where the maternal and paternal mutations were different were included in the study. Materials and Methods: Maternal peripheral blood was collected at different periods of gestation before the invasive fetal sampling procedure was done. The blood was centrifuged to isolate the plasma and prepare DNA. A size separation approach was used to isolate fetal DNA. Nested polymerase chain reaction (PCR-based protocols were developed for detection of the presence or absence of the paternal mutation. Results and Conclusions: There were 30 couples where the parental mutations were different. Of these, in 14 cases the paternal mutation was absent and in 16 cases it was present in the fetus. Using cell-free fetal DNA from maternal plasma, the absence of the paternal mutation was accurately determined in 12 of the 14 cases and the presence of the paternal mutation was correctly identified in 12 of the 16 cases. Thus, this non-invasive approach gave comparable results to those obtained by the conventional invasive fetal sampling methods in 24 cases giving an accuracy of 80.0%. Although the nested PCR approach enabled amplification of small quantities of cell-free DNA from maternal plasma at different periods of gestation after size separation to eliminate the more abundant maternal DNA, an accurate diagnosis of the presence or absence of the paternal mutation in the fetus was not possible in all cases to make it clinically

  5. Fetal Gene Therapy for Ornithine Transcarbamylase Deficiency by Intrahepatic Plasmid DNA-Micro-Bubble Injection Combined with Hepatic Ultrasound Insonation.

    Science.gov (United States)

    Oishi, Yoshie; Kakimoto, Takashi; Yuan, Wenji; Kuno, Shuichi; Yamashita, Hiromasa; Chiba, Toshio

    2016-06-01

    We evaluated the therapeutic efficacy of hepatic transfection of plasmid DNA using micro-bubbles and ultrasound insonation for fetal correction of ornithine transcarbamylase (OTC) deficiency in mice. Twenty-three sparse-fur heterozygous pregnant mice (day 16 of gestation) were divided into three groups: injection of plasmid-DNA micro-bubble mixture into fetal liver with ultrasound insonation (Tr, n = 11); control group 1 (C1), injection of plasmid-DNA micro-bubble mixture into fetal liver with no insonation (n = 5); and control group 2 (C2), injection of saline-micro-bubble mixture into fetal liver with ultrasound insonation (n = 7). Levels of blood ammonia and urinary orotic acid were significantly lower in the Tr group than in the C1 and C2 groups (p plasmid DNA transfection into fetal mouse liver, leading to one of the therapeutic methods in ammonia metabolism. This might provide more time for OTC-deficient infants until liver transplantation. PMID:26995155

  6. Dysregulation of the DNA Damage Response and KMT2A Rearrangement in Fetal Liver Hematopoietic Cells.

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    Mai Nanya

    Full Text Available Etoposide, a topoisomerase 2 (TOP2 inhibitor, is associated with the development of KMT2A (MLL-rearranged infant leukemia. An epidemiological study suggested that in utero exposure to TOP2 inhibitors may be involved in generation of KMT2A (MLL rearrangement. The present study examined the mechanism underlying the development of KMT2A (MLL-rearranged infant leukemia in response to in utero exposure to etoposide in a mouse model. Fetal liver hematopoietic stem cells were more susceptible to etoposide than maternal bone marrow mononuclear cells. Etoposide-induced Kmt2a breakage was detected in fetal liver hematopoietic stem cells using a newly developed chromatin immunoprecipitation (ChIP assay. Assessment of etoposide-induced chromosomal translocation by next-generation RNA sequencing (RNA-seq identified several chimeric fusion messenger RNAs that were generated by etoposide treatment. However, Kmt2a (Mll-rearranged fusion mRNA was detected in Atm-knockout mice, which are defective in the DNA damage response, but not in wild-type mice. The present findings suggest that in utero exposure to TOP2 inhibitors induces Kmt2a rearrangement when the DNA damage response is defective.

  7. A physiological delay in human fetal hemoglobin switching is associated with specific globin DNA hypomethylation.

    Science.gov (United States)

    Perrine, S P; Greene, M F; Cohen, R A; Faller, D V

    1988-02-01

    The human fetal-to-adult globin switch normally occurs on a fixed schedule, beginning at 32-34 weeks gestation, and recent studies have suggested an association between this developmental inactivation of the fetal (gamma) globin genes and the appearance of methylation within and around these genes. We have studied a population of infants in whom this switch does not occur before birth (infants of diabetic mothers, IDM) and examined the patterns of methylation surrounding their active gamma-globin genes, in comparison to the gamma-globin genes of age-matched controls who have switched their pattern of globin gene expression on schedule. All genomic DNA samples from infants with delays in the globin switch demonstrated extensive hypomethylation in the region of the gamma-globin genes, comparable to that found in the genomes of fetuses of less than 21 weeks gestation. DNA from the erythroid cells of infants of 32-40 weeks gestation had no detectable hypomethylation in the gamma-globin region. These findings support the concept that hypomethylation is an accurate developmental marker of globin gene switching, and suggest that globin gene expression in IDM may be arrested at an early preswitch stage. PMID:2449361

  8. Improvement in fetal DNA extraction from maternal plasma. Evaluation of the NucliSens Magnetic Extraction system and the QIAamp DSP Virus Kit in comparison with the QIAamp DNA Blood Mini Kit

    DEFF Research Database (Denmark)

    Clausen, Frederik Banch; Krog, Grethe Risum; Rieneck, Klaus;

    2007-01-01

    Prenatal diagnostic assays have been developed using free fetal DNA circulating in the maternal blood of pregnant women. Efficient DNA extraction is crucial for a robust analysis. To improve fetal DNA yield, we tested two manual extraction methods--the NucliSens Magnetic Extraction (NMAG) system...

  9. DNA fragmentation: manifestation of target cell destruction mediated by cytotoxic T-cell lines, lymphotoxin-secreting helper T-cell clones, and cell-free lymphotoxin-containing supernatant

    International Nuclear Information System (INIS)

    A Lyt-2+, trinitrophenyl-specific, lymphotoxin-secreting, cytotoxic T-cell line, PCl 55, mediates the digestion of target cell DNA into discretely sized fragments. This phenomenon manifests itself within 30 min after effector cell encounter as measured by the release of 3H counts from target cells prelabeled with [3H]deoxythymidine and occurs even at very low effector to target cell ratios (0.25:1). A Lyt-1+, ovalbumin-specific, lymphotoxin-secreting T-helper cell clone, 5.9.24, is also able to mediate fragmentation of target cell DNA over a time course essentially indistinguishable from the cytotoxic T lymphocyte-mediated hit. Cell-free lymphotoxin-containing supernatants also cause release of DNA from targets, although they require a longer time course, on the order of 24 hr. In contrast, lysis of cells by antibody plus complement or Triton X-100 does not result in DNA release even after extended periods of incubation (24 hr). All three treatments that result in the release of DNA from cells cause fragmentation of that DNA into discretely sized pieces that are multiples of 200 base pairs. The results thus suggest that cytotoxic T cells, lymphotoxin-secreting helper clones with cytolytic activity, and lymphotoxin all effect target cell destruction by means of a similar mechanism and that observed differences in time course and the absence of target cell specificity in killing mediated by lymphotoxin may simply reflect differences in the mode of toxin delivery

  10. Quantification of Cell-free HER-2 DNA in Plasma from Breast Cancer Patients: Sensitivity for Detection of Metastatic Recurrence and Gene Amplification

    Directory of Open Access Journals (Sweden)

    Patricia Diana Sørensen

    2015-08-01

    Conclusion: Amplified HER-2 DNA can be detected in plasma when using a ratio between cfHER-2 DNA and a reference gene. cfHER-2 DNA could not be used to dis‐ criminate between patients with primary breast cancer and healthy controls, and could not predict the development of metastatic disease.

  11. Analysis of expressed sequence tags from a fetal human heart cDNA library

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, D.M.; Fung, Y.W.; Wang, R.X.; Laurenssen, C.M. [Univ. of Toronto, Ontario (Canada)] [and others

    1995-11-20

    Single-pass sequencing of randomly selected cDNA clones to generate expressed sequence tags (ESTs) has been widely used to identify novel genes and to study gene expression in a variety of tissues. We have generated 2244 ESTs from a human fetal heart library (Gen-Bank Accession Nos. R30692-30774 and R56965-58824), which we present in this report. Of these, 51.7% showed no homology to known genes or were similar only to other ESTs, while 48.4% demonstrated homology to known transcripts. A total of 764 ESTs corresponding to known genes were used to study gene expression patterns in the fetal heart and to analyze differences in these patterns from those observed in the adult heart. These analyses demonstrate the utility of ESTs and sequence-tagged clones in comparative studies of gene expression in the cardiovascular system, and they reveal that differential gene expression underlies the structural and functional characteristics of the developing heart. 48 refs., 1 fig., 3 tabs.

  12. A genome-wide association study identifies UGT1A1 as a regulator of serum cell-free DNA in young adults: The Cardiovascular Risk in Young Finns Study.

    Directory of Open Access Journals (Sweden)

    Juulia Jylhävä

    Full Text Available INTRODUCTION: Circulating cell-free DNA (cf-DNA is a useful indicator of cell death, and it can also be used to predict outcomes in various clinical disorders. Several innate immune mechanisms are known to be involved in eliminating DNA and chromatin-related material as part of the inhibition of potentially harmful autoimmune responses. However, the exact molecular mechanism underlying the clearance of circulating cf-DNA is currently unclear. METHODS: To examine the mechanisms controlling serum levels of cf-DNA, we carried out a genome-wide association analysis (GWA in a cohort of young adults (aged 24-39 years; n = 1841; 1018 women and 823 men participating in the Cardiovascular Risk in Young Finns Study. Genotyping was performed with a custom-built Illumina Human 670 k BeadChip. The Quant-iT(TM high sensitivity DNA assay was used to measure cf-DNA directly from serum. RESULTS: The results revealed that 110 single nucleotide polymorphisms (SNPs were associated with serum cf-DNA with genome-wide significance (p<5×10(-8. All of these significant SNPs were localised to chromosome 2q37, near the UDP-glucuronosyltransferase 1 (UGT1 family locus, and the most significant SNPs localised within the UGT1 polypeptide A1 (UGT1A1 gene region. CONCLUSION: The UGT1A1 enzyme catalyses the detoxification of several drugs and the turnover of many xenobiotic and endogenous compounds by glucuronidating its substrates. These data indicate that UGT1A1-associated processes are also involved in the regulation of serum cf-DNA concentrations.

  13. Structure-Activity Relationships for DNA Damage by Alkenylbenzenes in Turkey Egg Fetal Liver.

    Science.gov (United States)

    Kobets, Tetyana; Duan, Jian-Dong; Brunnemann, Klaus D; Etter, Sylvain; Smith, Benjamin; Williams, Gary M

    2016-04-01

    Certain alkenylbenzenes (AB), flavoring chemicals naturally occurring in spices and herbs, are established to be cytotoxic and hepatocarcinogenic in rodents. The purpose of the present study was to determine the DNA damaging potential of key representatives of this class using the Turkey Egg Genotoxicity Assay. Medium white turkey eggs with 22- to 24-day-old fetuses received three injections of nine AB with different carcinogenic potentials: safrole (1, 2 mg/egg), methyl eugenol (2, 4 mg/egg), estragole (20, 40 mg/egg), myristicin (25, 50 mg/egg), elemicin (20, 50 mg/egg), anethole (5, 10 mg/egg), methyl isoeugenol (40, 80 mg/egg), eugenol (1, 2.5 mg/egg), and isoeugenol (1, 4 mg/egg). Three hours after the last injection, fetal livers were harvested for measurement of DNA strand breaks, using the comet assay and DNA adducts formation, using the nucleotide(3) (2)P-postlabeling assay. Estragole, myristicin, and elemicin induced DNA stand breaks. These compounds as well as safrole, methyl eugenol and anethole, at the highest doses tested, induced DNA adduct formation. Methyl isoeugenol, eugenol, and isoeugenol did not induce genotoxicity. The genotoxic AB all had the structural features of either a double bond in the alkenyl side chain at the terminal 2',3'-position, favorable to formation of proximate carcinogenic 1'-hydroxymetabolite or terminal epoxide, or the absence of a free phenolic hydroxyl group crucial for formation of a nontoxic glucuronide conjugate. In contrast, methyl isoeugenol, eugenol and isoeugenol, which were nongenotoxic, possessed chemical features, unfavorable to activation. PMID:26719370

  14. Identification of Human N-Myristoylated Proteins from Human Complementary DNA Resources by Cell-Free and Cellular Metabolic Labeling Analyses

    OpenAIRE

    Takamitsu, Emi; Otsuka, Motoaki; Haebara, Tatsuki; YANO, Manami; Matsuzaki, Kanako; Kobuchi, Hirotsugu; Moriya, Koko; Utsumi, Toshihiko

    2015-01-01

    To identify physiologically important human N-myristoylated proteins, 90 cDNA clones predicted to encode human N-myristoylated proteins were selected from a human cDNA resource (4,369 Kazusa ORFeome project human cDNA clones) by two bioinformatic N-myristoylation prediction systems, NMT-The MYR Predictor and Myristoylator. After database searches to exclude known human N-myristoylated proteins, 37 cDNA clones were selected as potential human N-myristoylated proteins. The susceptibility of the...

  15. Neither DNA hypomethylation nor changes in the kinetics of erythroid differentiation explain 5-azacytidine's ability to induce human fetal hemoglobin

    OpenAIRE

    Mabaera, Rodwell; Greene, Michael R.; Richardson, Christine A.; Conine, Sarah J; Kozul, Courtney D.; Lowrey, Christopher H.

    2008-01-01

    5-azacytidine (5-Aza) is a potent inducer of fetal hemoglobin (HbF) in people with β-thalassemia and sickle cell disease. Two models have been proposed to explain this activity. The first is based on the drug's ability to inhibit global DNA methylation, including the fetal globin genes, resulting in their activation. The second is based on 5-Aza's cytotoxicity and observations that HbF production is enhanced during marrow recovery. We tested these models using human primary cells in an in vit...

  16. Haplotype-based approach for noninvasive prenatal tests of Duchenne muscular dystrophy using cell-free fetal DNA in maternal plasma

    DEFF Research Database (Denmark)

    Xu, Yan; Li, Xuchao; Ge, Hui-Juan;

    2015-01-01

    Purpose:This study demonstrates noninvasive prenatal testing (NIPT) for Duchenne muscular dystrophy (DMD) using a newly developed haplotype-based approach.Methods:Eight families at risk for DMD were recruited for this study. Parental haplotypes were constructed using target-region sequencing data....... With a mean depth of 30× achieved in the 10-Mb target region of each sample, the noninvasive results were consistent with those of the invasive procedure.Conclusion:This is the first report of NIPT for DMD and the first application of a haplotype-based approach in NIPT for X-linked diseases. With further...... improvements in accuracy, this haplotype-based strategy could be feasible for NIPT for DMD and even other X-linked single-gene disorders.Genet Med advance online publication 05 February 2015Genetics in Medicine (2015); doi:10.1038/gim.2014.207....

  17. Immune Reactivation by Cell-Free Fetal DNA in Healthy Pregnancies Re-Purposed to Target Tumors: Novel Checkpoint Inhibition in Cancer Therapeutics

    OpenAIRE

    Enninga, Elizabeth Ann L.; Nevala, Wendy K.; Holtan, Shernan G.; Svetomir N. Markovic

    2015-01-01

    The role of the immune system in cancer progression has become increasingly evident over the past decade. Chronic inflammation in the promotion of tumorigenesis is well established, and cancer-associated tolerance/immune evasion has long been appreciated. Recent developments of immunotherapies targeting cancer-associated inflammation and immune tolerance, such as cancer vaccines, cell therapies, neutralizing antibodies, and immune checkpoint inhibitors, have shown promising clinical results. ...

  18. Non-invasive prenatal molecular detection of a fetal point mutation for congenital adrenal hyperplasia using co-amplification at lower denaturation temperature PCR

    Institute of Scientific and Technical Information of China (English)

    DU Juan; ZOU Xin; PAN Yi; LI Shuang-fei; LU Guang-xiu

    2010-01-01

    @@ Conventional prenatal diagnosis relies on invasive chorionic biopsy or amniocentesis, which increases the risk of miscarriage, and is undertaken at 11-20 weeks gestation.1 The discovery of cell-free fetal DNA in maternal plasma has, however, offered a new strategy for non-invasive prenatal diagnosis.2

  19. Fetal gender determination through Y-STR analysis of maternal plasma during the third trimester of pregnancy

    OpenAIRE

    Hanaa M.H. Aal-Hamdan; Ahmed M. Refaat; Saranya R. Babu; Abdul Rauf Choudhry

    2015-01-01

    Background: The passage of nucleated cells between mother and fetus is well recognized (Lo et al., 1989, 1996). As well as, cell-free fetal DNA in maternal plasma or serum is at present widely investigated as a source of fetal genetic material (Stanghellini et al., 2006) [18]. There has been much recent interest in the use of DNA derived from plasma or serum (Boland, 1996). This DNA can be utilized for molecular diagnosis as well as prenatal sex discernment. Objective: To establish an easy...

  20. Diagnostic Validity of Serum and Peritoneal TNF-alpha, high sensitivity CRP and Plasma Cell-Free Nuclear DNA (ccf nDNA as Biomarkers of Pelvic Endometriosis- A Case Control Study

    Directory of Open Access Journals (Sweden)

    Ahmed F Koura, MSc*, Mohamed A Yehia, Waleed H ElTantawy, Adel S Salah El Din, Dina El-Sayed ElShennawy

    2013-04-01

    Full Text Available Introduction: Endometriosis is a disease defined by the presence of endometrial glands and stroma located outside the uterine cavity. These ectopic implants can be found throughout the pelvis, on and within the ovaries, abutting the uterine ligaments, occupying the rectovaginal septum, invading the intestinal serosa, and along the parietal peritoneum. Endometrial implantation at distant sites such as the pleura, lung, within surgical scars, and along the diaphragm also has been reported. (1. It results often in subfertility and pain, occurs mainly in women of reproductive age (16–50 years and has a progressive character in at least 50%, but the rate and risk factors for progression are unknown. Endometriosis can be classified into four stages: minimal, mild, moderate and severe. The gold standard for the diagnosis of endometriosis is laparoscopic inspection, ideally with histological confirmation. (2, however, is an invasive technique and should be performed only after imaging techniques prove insufficient for confident diagnosis. (3 Lack of a non-invasive diagnostic test contributes to the long delay between onset of symptoms and diagnosis of endometriosis. (2 Additional tools are needed for non-invasive classifications in order to reduce the number of unnecessary laparoscopies without adversely affecting outcomes. Finding specific and more sensitive biomarkers in endometriosis is critical, because endometriosis is usually diagnosed only in advanced stages, and there is a high rate of morbidity for this disease. (4 Aim of the work: The aim of the current study is to assess the validity of serum and peritoneal high sensitivity CRP and TNF-alpha and plasma cell-free nuclear DNA (ccf nDNA as biomarkers in early diagnosis of pelvic endometriosis.Methods: This study was conducted at the Obstetrics & Gynecology department, Maternity Hospital, Ain Shams University. This is a case control study of 120 women scheduled for diagnostic laparoscopy

  1. Determinação pré-natal do sexo fetal por meio da análise de DNA no plasma materno Prenatal fetal gender determination by analysis of DNA from maternal pasma

    Directory of Open Access Journals (Sweden)

    José Eduardo Levi

    2003-01-01

    Full Text Available OBJETIVO: avaliar um novo método de determinação do sexo fetal pela análise de DNA obtido do plasma materno. MÉTODOS: sangue periférico (10 mL foi coletado de mulheres grávidas em diferentes idades gestacionais. O plasma foi separado e o DNA isolado do mesmo foi submetido à reação em cadeia da polimerase (PCR com oligonucleotídeos iniciadores derivados do gene DYS14 específico do cromossomo Y. RESULTADOS: foram analisadas amostras de 212 pacientes. O resultado da PCR foi comparado ao sexo determinado pela ultra-sonografia e/ou do nascimento. Houve concordância em 209 das 212 pacientes. Nos 3 casos discordantes a PCR apontou resultado feminino, sendo as três amostras coletadas antes da 8ª semana de gravidez. CONCLUSÃO: o método de PCR desenvolvido para a determinação do sexo fetal possui excelente sensibilidade e especificidade, permitindo seu uso rotineiro. O resultado de sexo masculino possui maior confiabilidade que o feminino, principalmente em idade gestacional precoce. Novas aplicações para o DNA fetal no plasma materno estão sendo pesquisadas, permitindo no futuro o diagnóstico não invasivo de uma série de doenças.PURPOSE: to evaluate a new method of fetal sex determination through the analysis of DNA from maternal plasma. METHODS: peripheral blood (10 mL was drawn from pregnant women in different gestational ages. Plasma was separated and isolated DNA was submitted to the polymerase chain reaction (PCR with primers from the Y-chromosome-specific gene DYS14. RESULTS: two hundred and twelve patients were enrolled. PCR results were compared to either confirmatory ultrasonography and/or birth. Agreement was observed in 209 samples. Discordant plasmas had a PCR result of female fetus and all of them were derived from mothers with less than 8 weeks of pregnancy. CONCLUSION: the PCR method developed for fetal sex determination has excellent sensitivity and specificity, permitting its use as a routine test. A PCR result

  2. Increased DNA methylation of scavenger receptor class B type I contributes to inhibitory effects of prenatal caffeine ingestion on cholesterol uptake and steroidogenesis in fetal adrenals

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Dong-Mei; He, Zheng; Ma, Liang-Peng; Wang, Lin-Long [Department of Pharmacology, Wuhan University School of Basic Medical Sciences, Wuhan 430071 (China); Ping, Jie, E-mail: pingjie@whu.edu.cn [Department of Pharmacology, Wuhan University School of Basic Medical Sciences, Wuhan 430071 (China); Hubei Provincial Key Laboratory of Developmentally Originated Diseases, Wuhan 430071 (China); Research Center of Food and Drug Evaluation, Wuhan University, Wuhan 430071 (China); Wang, Hui [Department of Pharmacology, Wuhan University School of Basic Medical Sciences, Wuhan 430071 (China); Hubei Provincial Key Laboratory of Developmentally Originated Diseases, Wuhan 430071 (China); Research Center of Food and Drug Evaluation, Wuhan University, Wuhan 430071 (China)

    2015-06-01

    Steroid hormones synthesized from cholesterol in the fetal adrenal are crucial for fetal development. We have observed the inhibited fetal adrenal corticosterone synthesis and increased intrauterine growth retardation (IUGR) rate in rats under prenatal caffeine ingestion. The aim of this study is to evaluate the effects of prenatal caffeine ingestion on cholesterol supply in fetal adrenal steroidogenesis in rats and explore the underlying epigenetic mechanisms. Pregnant Wistar rats were treated with 60 mg/kg·d caffeine from gestational day (GD) 7 to GD17. Histological changes of fetal adrenals and increased IUGR rates were observed in the caffeine group. There were significantly decreased steroid hormone contents and cholesterol supply in caffeine-treated fetal adrenals. Data from the gene expression array suggested that prenatal caffeine ingestion caused increased expression of genes related to DNA methylation and decreased expression of genes related to cholesterol uptake. The following conjoint analysis of DNA methylation array with these differentially expressed genes suggested that scavenger receptor class B type I (SR-BI) may play an important role in caffeine-induced cholesterol supply deficiency. Moreover, real-time RT-PCR and immunohistochemical detection certified the inhibitory effects of caffeine on both mRNA expression and protein expression of SR-BI in the fetal adrenal. And the increased DNA methylation frequency in the proximal promoter of SR-BI was confirmed by bisulfite-sequencing PCR. In conclusion, prenatal caffeine ingestion can induce DNA hypermethylation of the SR-BI promoter in the rat fetal adrenal. These effects may lead to decreased SR-BI expression and cholesterol uptake, which inhibits steroidogenesis in the fetal adrenal. - Highlights: • Prenatal caffeine ingestion inhibits steroid hormone production in the fetal adrenal. • Prenatal caffeine ingestion inhibits cholesterol uptake in the fetal adrenal. • Prenatal caffeine

  3. Increased DNA methylation of scavenger receptor class B type I contributes to inhibitory effects of prenatal caffeine ingestion on cholesterol uptake and steroidogenesis in fetal adrenals

    International Nuclear Information System (INIS)

    Steroid hormones synthesized from cholesterol in the fetal adrenal are crucial for fetal development. We have observed the inhibited fetal adrenal corticosterone synthesis and increased intrauterine growth retardation (IUGR) rate in rats under prenatal caffeine ingestion. The aim of this study is to evaluate the effects of prenatal caffeine ingestion on cholesterol supply in fetal adrenal steroidogenesis in rats and explore the underlying epigenetic mechanisms. Pregnant Wistar rats were treated with 60 mg/kg·d caffeine from gestational day (GD) 7 to GD17. Histological changes of fetal adrenals and increased IUGR rates were observed in the caffeine group. There were significantly decreased steroid hormone contents and cholesterol supply in caffeine-treated fetal adrenals. Data from the gene expression array suggested that prenatal caffeine ingestion caused increased expression of genes related to DNA methylation and decreased expression of genes related to cholesterol uptake. The following conjoint analysis of DNA methylation array with these differentially expressed genes suggested that scavenger receptor class B type I (SR-BI) may play an important role in caffeine-induced cholesterol supply deficiency. Moreover, real-time RT-PCR and immunohistochemical detection certified the inhibitory effects of caffeine on both mRNA expression and protein expression of SR-BI in the fetal adrenal. And the increased DNA methylation frequency in the proximal promoter of SR-BI was confirmed by bisulfite-sequencing PCR. In conclusion, prenatal caffeine ingestion can induce DNA hypermethylation of the SR-BI promoter in the rat fetal adrenal. These effects may lead to decreased SR-BI expression and cholesterol uptake, which inhibits steroidogenesis in the fetal adrenal. - Highlights: • Prenatal caffeine ingestion inhibits steroid hormone production in the fetal adrenal. • Prenatal caffeine ingestion inhibits cholesterol uptake in the fetal adrenal. • Prenatal caffeine

  4. Biophysical analysis of natural, double-helical DNA modified by dinuclear platinum(II) organometallic compound in a cell-free medium

    Czech Academy of Sciences Publication Activity Database

    Marini, Victoria; Kašpárková, Jana; Nováková, Olga; Scolaro, L. M.; Romeo, R.; Brabec, Viktor

    Florence, 2001, s. 9. [COST Action: D20 Metal Compounds in the Treatment of Cancer . Florence (IT), 29.08.2001] Institutional research plan: CEZ:AV0Z5004920 Keywords : DNA * platinum * cross-link Subject RIV: BO - Biophysics

  5. Quantification of Cell-free HER-2 DNA in Plasma from Breast Cancer Patients: Sensitivity for Detection of Metastatic Recurrence and Gene Amplification

    OpenAIRE

    Patricia Diana Sørensen; Rikke Fredslund Andersen; Niels Pallisgaard; Jonna Skov Madsen; Erik Hugger Jakobsen; Ivan Brandslund

    2015-01-01

    The purpose of this study was to quantify the free-circu‐ lating plasma HER-2 DNA (cfHER-2 DNA) and to assess the ability of analysis to discriminate between patients with primary breast cancer and healthy controls in order to detect metastatic recurrence in comparison with serum HER-2 protein and also HER-2 gene amplification. The study population consisted of 100 patients with primary breast cancer and 50 healthy female donors. An additional 22 patients with metastases were subsequently ...

  6. DNA repair enzyme deficiency and in vitro complementation of the enzyme activity in cell-free extracts from ataxia telangiectasia fibroblasts

    International Nuclear Information System (INIS)

    Three ataxia telangiectasia homozygotes, one heterozygote and normal fibroblast strains were compared as to the capacity of their cellular extracts to enhance the priming activity of gamma-irradiated colicin E1 DNA for purified DNA polymerase (EC 2.7.7.7) of Escherichia coli. It was found that homozygotes had substantially lower activity than normal strains, while no difference was detected between the heterozygote and normal strains. In vitro complementation of the activity occurred between extracts of certain strains of homozygotes, allocating them to two complementation groups. (Auth.)

  7. The correlation between cell-free DNA and tumour burden was estimated by PET/CT in patients with advanced NSCLC

    DEFF Research Database (Denmark)

    Nygaard, A D; Holdgaard, Paw; Spindler, K-L G;

    2014-01-01

    -FDG) PET/computed tomography (CT) scan was performed and evaluated in terms of metabolic tumour volume (MTV) and total lesion glycolysis (TLG). Tumour contours were delineated semi-automatically by a threshold standardised uptake value (SUV) of 2.5. The primary end point was correlation among cfDNA, MTV...

  8. Noninvasive fetal RhD genotyping.

    Science.gov (United States)

    Clausen, Frederik Banch; Damkjær, Merete Berthu; Dziegiel, Morten Hanefeld

    2014-04-01

    Immunization against RhD is the major cause of hemolytic disease of the fetus and newborn (HDFN), which causes fetal or neonatal death. The introduction of postnatal immune prophylaxis in the 1960s drastically reduced immunization incidents in pregnant, D-negative women. In several countries, antenatal prophylaxis is combined with postnatal prophylaxis to further minimize the immunization risk. Due to lack of knowledge of the fetal RhD type, antenatal prophylaxis is given to all D-negative women. In the European population, approximately 40% of pregnant women carry a D-negative fetus and are thus at no risk of immunization. Noninvasive fetal RhD genotyping enables antenatal prophylaxis to be targeted to only those women carrying a D-positive fetus to avoid unnecessary treatment. Based on an analysis of cell-free fetal DNA from the plasma of pregnant women, this approach has recently undergone technical improvements and rapid clinical implementation. As a screening assay, the sensitivity is >99.3% from a gestational age of approximately 10-11 weeks. In addition, fetal RhD genotyping is widely used to assess the risk of HDFN in anti-D immunized women. PMID:24642067

  9. Quantitative identification of mutant alleles derived from lung cancer in plasma cell-free DNA via anomaly detection using deep sequencing data.

    Directory of Open Access Journals (Sweden)

    Yoji Kukita

    Full Text Available The detection of rare mutants using next generation sequencing has considerable potential for diagnostic applications. Detecting circulating tumor DNA is the foremost application of this approach. The major obstacle to its use is the high read error rate of next-generation sequencers. Rather than increasing the accuracy of final sequences, we detected rare mutations using a semiconductor sequencer and a set of anomaly detection criteria based on a statistical model of the read error rate at each error position. Statistical models were deduced from sequence data from normal samples. We detected epidermal growth factor receptor (EGFR mutations in the plasma DNA of lung cancer patients. Single-pass deep sequencing (>100,000 reads was able to detect one activating mutant allele in 10,000 normal alleles. We confirmed the method using 22 prospective and 155 retrospective samples, mostly consisting of DNA purified from plasma. A temporal analysis suggested potential applications for disease management and for therapeutic decision making to select epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI.

  10. The study of responses to 'model' DNA breaks induced by restriction endonucleases in cells and cell-free systems: achievements and difficulties

    International Nuclear Information System (INIS)

    The use of restriction endonucleases (RE) as a means of implicating DNA double-strand breaks (dsb) in cellular responses is reviewed. The introduction of RE into cells leads to many of the responses known to be characteristic of radiation damage -cell killing, chromosomal aberration, oncogenic transformation, gene mutation and amplification. Additionally, radiosensitive cell lines are hypersensitive to RE, including those from the human disorder ataxia-telangiectasia. However, quantitation of response and comparisons of the effectiveness of different RE are difficult, partly because of unknown activity and lifetime of RE in the cell. Re-induced dsb have also been used to reveal molecular mechanisms of repair and misrepair at specific sites in DNA. Dsb have been implicated in recombination processes including those leading to illegitimate rejoining (formation of deletions and rearrangements) at short sequence features in DNA. Also model dsb act as a signal to activate other cellular processes, which may influence or indirectly cause some responses, including cell death. In these signalling responses the detailed chemistry at the break site may not be very important, perhaps explaining why there is considerable overlap in responses to RE and to ionizing radiations. (author)

  11. Diagnóstico prenatal no invasivo: Ácidos nucleicos de origen fetal en sangre materna Non invasive prenatal diagnosis: Fetal nucleic acid analysis in maternal blood

    Directory of Open Access Journals (Sweden)

    Carla Sesarini

    2010-12-01

    decade, the presence of cell-free fetal DNA in maternal blood has been identified. These fetal DNA fragments would derive from the placenta and are not detected after delivery, making them a source of fetal material for carrying out diagnosis techniques using maternal blood. However, the vast majority of cell free DNA in maternal circulation is of maternal origin, with the fetal component contributing from 3% to 6% and rising towards term. Available methodologies do not allow separation of fetal from maternal cell free DNA, so current applications have been focused on the analysis of genes not present in the mother, such as Y chromosome sequences, or RHD gene in RhD-negative women, or paternal or de novo mutations. Also, the detection of cell-free fetal RNA in maternal blood offers the possibility of obtaining information regarding genetic expression profiles of embrionic tissues, and using genes expressed only at the feto-placental unit, controls for the presence of fetal material could be established, regardless of maternal genetic tissue. The present article describes the evidences regarding the passage of fetal nucleic acids to maternal circulation, its current prenatal diagnosis application and possible future perspectives.

  12. Maternal diet during pregnancy induces gene expression and DNA methylation changes in fetal tissues in sheep

    Directory of Open Access Journals (Sweden)

    Xianyong eLan

    2013-04-01

    Full Text Available Studies in rats and mice have established that maternal nutrition induces epigenetic modifications, sometimes permanently, that alter gene expression in the fetus, which in turn leads to phenotypic changes. However, limited data is available on the influence of maternal diet on epigenetic modifications and gene expression in sheep. Therefore, the objectives of this study were to investigate the impact of different maternal dietary energy sources on the expression of imprinted genes in fetuses in sheep. Ewes were naturally bred to a single sire and from d 67 ± 3 of gestation until necropsy (d 130 ± 1, they were fed one of three diets of alfalfa haylage (HY; fiber, corn (CN; starch, or dried corn distiller’s grains (DG; fiber plus protein plus fat. A total of 26 fetuses were removed from the dams and longissimus dorsi, semitendinosus, perirenal adipose depot, and subcutaneous adipose depot tissues were collected for expression and DNA methylation analyses. Expression analysis of nine imprinted genes and three DNA methylatransferase (DNMTs genes showed significant effects of the different maternal diets on the expression of these genes. The methylation levels of CpG islands of both IGF2R and H19 were higher in HY and DG than CN fetuses in both males and females. This result is consistent with the low amino acid content of the CN diet, a source of methyl group donors, compared to HY and DG diets. Thus, results of this study provide evidence of association between maternal nutrition during pregnancy and transcriptomic and epigenomic alterations of imprinted genes and DNMTs in the fetal tissues.

  13. Digital PCR analysis of plasma cell-free DNA for non-invasive detection of drug resistance mechanisms in EGFR mutant NSCLC: Correlation with paired tumor samples

    Science.gov (United States)

    Ishii, Hidenobu; Azuma, Koichi; Sakai, Kazuko; Kawahara, Akihiko; Yamada, Kazuhiko; Tokito, Takaaki; Okamoto, Isamu; Nishio, Kazuto; Hoshino, Tomoaki

    2015-01-01

    As the development of resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has become an issue of concern, identification of the mechanisms responsible has become an urgent priority. However, for research purposes, it is not easy to obtain tumor samples from patients with EGFR mutation-positive non-small-cell lung cancer (NSCLC) that has relapsed after treatment with EGFR-TKIs. Here, using digital PCR assay as an alternative and noninvasive method, we examined plasma and tumor samples from patients with relapsed NSCLC to establish the inter-relationships existing among T790M mutation, activating EGFR mutations, HER2 amplification, and MET amplification. Paired samples of tumor and blood were obtained from a total of 18 patients with NSCLC after they had developed resistance to EGFR-TKI treatment, and the mechanisms of resistance were analyzed by digital PCR. Digital PCR analysis of T790M mutation in plasma had a sensitivity of 81.8% and specificity of 85.7%, the overall concordance between plasma and tissue samples being 83.3%. MET gene copy number gain in tumor DNA was observed by digital PCR in three patients, of whom one exhibited positivity for MET amplification by FISH, whereas no patient demonstrated MET and HER2 copy number gain in plasma DNA. Digital PCR analysis of plasma is feasible and accurate for detection of T790M mutation in NSCLC that becomes resistant to treatment with EGFR-TKIs. PMID:26334838

  14. Noninvasive Detection of Fetal Subchromosome Abnormalities via Deep Sequencing of Maternal Plasma

    OpenAIRE

    Srinivasan, Anupama; Bianchi, Diana W.; Huang, Hui; Sehnert, Amy J.; Rava, Richard P.

    2013-01-01

    The purpose of this study was to determine the deep sequencing and analytic conditions needed to detect fetal subchromosome abnormalities across the genome from a maternal blood sample. Cell-free (cf) DNA was isolated from the plasma of 11 pregnant women carrying fetuses with subchromosomal duplications and deletions, translocations, mosaicism, and trisomy 20 diagnosed by metaphase karyotype. Massively parallel sequencing (MPS) was performed with 25-mer tags at approximately 109 tags per samp...

  15. Modulation of DNA double-strand break repair activity in cell-free extracts of gamma-irradiated mouse testicular cells

    International Nuclear Information System (INIS)

    DNA double-strand breaks (DSBs) are potentially mutagenic lesions demanding effective damage recognition and repair. Even a single DSB can be detrimental if left unrepaired or misrepaired, and if present in gamete, it can cause foetal wastage or malformations/congenital defects in the offspring. The threats posed by DSBs have triggered the evolution of two major pathways of DSB repair, homologous recombination-mediated repair (HRR) and non-homologous end-joining (NHEJ), conserved from bacteria to mammals. Though HRR is more predominant in bacteria and yeast, NHEJ is more efficient in mammalian somatic cells. Studies in our laboratory have shown that both the pathways are equally efficient in mammalian male germ cells

  16. Fetal sex determination in the first trimester of pregnancy using a Y chromosome-specific DNA probe

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Y.; Huang, S.; Chen, M.; Huang, Y.; Zhang, M.; Dong, J.; Ku, A.; Xu, S.

    1987-05-01

    Prenatal determination of fetal sex is important for the prevention of X-linked disorders such as hemophilia, Lesch-Nyhan syndrome and Duchenne muscular dystrophy. The complex procedures of prenatal diagnosis for X-linked disorders are unnecessary if the fetus is female, because usually no clinical symptoms ever appear in female. pY 3.4 probe used in this work for sex determination is a 3.4 kilobase human repeat sequence. The probe is specific for the Y chromosome of males and can be used for sex determination. The other prove pBLUR used in this paper as control is a widely dispersed, highly repeated human Alu family DNA sequence, represented equally in male and female DNA. On the basis of the relative densities of the autoradiographic spots produced by hybridization of fetal DNA with pY3.4 and pBLUR, the sex of fetus can be clearly identified. Further the authors can determine the radioactive intensity (cpm) of the hybridized DNA spots and the ratio of hybridization with Y3.4 to pBLUR (Y3.4/pBLUR x 10). Results show that the hybridization ratio of DNA from chorionic villi of male (1.03 +/- 0.24) is significantly higher than that of female (0.16 +/- 0.09). Therefore, sex determination of the fetus can be made, based on the ratio of pY3.4/pBLUR x 10. If necessary they can also use Southern hybridization with pY 3.4 probe of DNA isolated from chorionic villi to confirm the result of dot hybridization.

  17. Detection of fetal-specific DNA after enrichment for trophoblasts using the monoclonal antibody LK26 in model systems but failure to demonstrate fetal DNA in maternal peripheral blood

    DEFF Research Database (Denmark)

    Hviid, T V; Sørensen, S; Morling, N

    1999-01-01

    Trophoblast cells can be detected in maternal blood during normal human pregnancy and DNA from these cells may be used for non-invasive prenatal diagnosis of inherited diseases. The possibility of enriching trophoblast cells from maternal blood samples using a monoclonal antibody (LK26) against a...... peripheral maternal blood samples. However, it was not possible to detect fetal DNA sequences in these samples, most probably dueto the extremely low number of trophoblast cells. Positive identification and retrieval of trophoblast cells in suspension or trophoblast nuclear material prepared on microscope...... chain reaction (PCR) and automated fluorescence-based genotyping. After successful initial experiments using retroplacental blood samples with a high number of trophoblast cells or an artificial mixture of trophoblast cells and blood, several versions of the enrichment method were attempted on...

  18. Detection of fetal-specific DNA after enrichment for trophoblasts using the monoclonal antibody LK26 in model systems but failure to demonstrate fetal DNA in maternal peripheral blood

    DEFF Research Database (Denmark)

    Hviid, T V; Sørensen, S; Morling, N

    1999-01-01

    Trophoblast cells can be detected in maternal blood during normal human pregnancy and DNA from these cells may be used for non-invasive prenatal diagnosis of inherited diseases. The possibility of enriching trophoblast cells from maternal blood samples using a monoclonal antibody (LK26) against a...... peripheral maternal blood samples. However, it was not possible to detect fetal DNA sequences in these samples, most probably due to the extremely low number of trophoblast cells. Positive identification and retrieval of trophoblast cells in suspension or trophoblast nuclear material prepared on microscope...... chain reaction (PCR) and automated fluorescence-based genotyping. After successful initial experiments using retroplacental blood samples with a high number of trophoblast cells or an artificial mixture of trophoblast cells and blood, several versions of the enrichment method were attempted on...

  19. Sensitivity of Noninvasive Prenatal Detection of Fetal Aneuploidy from Maternal Plasma Using Shotgun Sequencing Is Limited Only by Counting Statistics

    OpenAIRE

    Fan, H. Christina; Quake, Stephen R.

    2010-01-01

    We recently demonstrated noninvasive detection of fetal aneuploidy by shotgun sequencing cell-free DNA in maternal plasma using next-generation high throughput sequencer. However, GC bias introduced by the sequencer placed a practical limit on the sensitivity of aneuploidy detection. In this study, we describe a method to computationally remove GC bias in short read sequencing data by applying weight to each sequenced read based on local genomic GC content. We show that sensitivity is limited...

  20. Translation in cell-free systems

    International Nuclear Information System (INIS)

    The simplest, unambiguous identification of a particular mRNA is the identification of its protein product. This can be established by translation of the mRNA of interest in a cell-free protein-synthesizing system. Messenger RNA protein product identification is important in the isolation of a particular mRNA species for cDNA cloning and in the identification of positive cDNA clones. The two high-activity translation systems in common use are those prepared from rabbit reticulocytes and from wheat germ. Both systems are easy to prepare, and both are available commercially. Each has advantages and disadvantages over the other and a choice between the two will depend on the type of mRNAs to be translated, the prejudices of experience, and availability. The main disadvantage of the reticulocyte system is that it requires removal of endogenous mRNA. However, this is a relatively simple procedure. The wheat germ system does not require removal of endogenous mRNA and may translate weakly initiating mRNAs more efficiently. However, ionic optima for translation in the wheat germ system are more sensitive to the nature and concentration of mRNA and may need to be determined for each template. The biggest problem with the use of the wheat germ system is its tendency to produce incomplete translation products due to premature termination

  1. 孕妇血清中游离胎儿DNA的分离及鉴定%Isolation and Identification of Free Fetal DNA in Maternal Serum

    Institute of Scientific and Technical Information of China (English)

    罗衡丽; 韦四喜; 刘丽荣; 赵鲁强; 张小蕾

    2011-01-01

    目的:建立一种稳定的提取母体血清DNA和鉴定胎儿DNA的方法.方法:采用裂解液煮沸法提取孕早、中、后期共90例孕妇血清DNA,应用PCR技术及亲子鉴定原理检测孕妇血清及夫妇双方DYZ1基因、DXS6804、DXS6799和D18S51以确认母体血清中胎儿DNA.结果:男胎孕妇血清中DNA DYZ1基因早、中、晚期的检出率分别为75%、88.89%、100%;血清中胎儿DNA的DXS6804、DXS6799、D18S51的孕早、中、后期检出率分别是76.67%、93.33%、100%.结论:裂解液煮沸法能提取孕妇血清游离DNA,且简便、快捷、提取效率高,可用于后续产前诊断;孕妇血清中存在胎儿游离DNA,并随孕周的增加而明显增加;STR位点扩增及亲子鉴定方法的应用,弥补了DYZ1序列仅能用于鉴定男性胎儿DNA的不足,可作为一种鉴定胎儿DNA的更为可靠的方法;X-STR(DXS6804、DXS6799)和Y染色体上的DYZ1位点可用于胎儿性别的鉴定,为性连锁遗传病的早期筛选提供了一种新的方法.%Objective: To establish a stable method for isolation and identification of fetal DNA in maternal serum. Methods: Serum DNA of 90 pregnant women was isolated with boiling lysis method.PCR was used to detect DYZ1 gene, and short tandem repeat sequences (STR) of DXS6804,DXS6799 and D18S51 in serum of couple. Fetal DNA sequences were identified following paternity test principle. Results: Detecting rates of male fetal DYZ1 gene in early, intermediate and late stages were 75%, 88. 89%, and 100% respectively. Detecting rates of fetal DNA sequences of DXS6804,DXS6799, and D18S51 in early, intermediate and late stages were 76.67%, 93.33%, and 100% respectively. Conclusions: Fetal DNA exists in maternal serum and its concentration increases significantly as gestational age increased. Free DNA in maternal serum could be easily and efficiently isolated with boiling lysis method and could be used in following prenatal diagnosis. STR loci amplification and

  2. Cisplatin-DNA adduct formation in rat spermatozoa and its effect on fetal development

    NARCIS (Netherlands)

    Hooser, S.T.; Dijk-Knijnenburg, C.M. van; Waalkens-Berendsen, I.D.H.; Smits-van Prooije, A.E.; Snoeij, N.J.; Baan, R.A.; Fichtinger-Schepman, M.J.

    2000-01-01

    Exposure of males to some genotoxic chemicals causes DNA damage in spermatozoa resulting in embryotoxicity and developmental defects in their offspring. This study demonstrates that cisplatin-DNA adducts could be measured in spermatozoa following treatment with the antineoplastic drug, cisplatin. Th

  3. Flexible Programming of Cell-Free Protein Synthesis Using Magnetic Bead-Immobilized Plasmids

    OpenAIRE

    Lee, Ka-Young; Lee, Kyung-Ho; Park, Ji-Woong; Kim, Dong-Myung

    2012-01-01

    The use of magnetic bead-immobilized DNA as movable template for cell-free protein synthesis has been investigated. Magnetic microbeads containing chemically conjugated plasmids were used to direct cell-free protein synthesis, so that protein generation could be readily programmed, reset and reprogrammed. Protein synthesis by using this approach could be ON/OFF-controlled through repeated addition and removal of the microbead-conjugated DNA and employed in sequential expression of different g...

  4. An Optimized Method for Accurate Fetal Sex Prediction and Sex Chromosome Aneuploidy Detection in Non-Invasive Prenatal Testing

    Science.gov (United States)

    Li, Haibo; Ding, Jie; Wen, Ping; Zhang, Qin; Xiang, Jingjing; Li, Qiong; Xuan, Liming; Kong, Lingyin; Mao, Yan; Zhu, Yijun; Shen, Jingjing; Liang, Bo; Li, Hong

    2016-01-01

    Massively parallel sequencing (MPS) combined with bioinformatic analysis has been widely applied to detect fetal chromosomal aneuploidies such as trisomy 21, 18, 13 and sex chromosome aneuploidies (SCAs) by sequencing cell-free fetal DNA (cffDNA) from maternal plasma, so-called non-invasive prenatal testing (NIPT). However, many technical challenges, such as dependency on correct fetal sex prediction, large variations of chromosome Y measurement and high sensitivity to random reads mapping, may result in higher false negative rate (FNR) and false positive rate (FPR) in fetal sex prediction as well as in SCAs detection. Here, we developed an optimized method to improve the accuracy of the current method by filtering out randomly mapped reads in six specific regions of the Y chromosome. The method reduces the FNR and FPR of fetal sex prediction from nearly 1% to 0.01% and 0.06%, respectively and works robustly under conditions of low fetal DNA concentration (1%) in testing and simulation of 92 samples. The optimized method was further confirmed by large scale testing (1590 samples), suggesting that it is reliable and robust enough for clinical testing. PMID:27441628

  5. An Optimized Method for Accurate Fetal Sex Prediction and Sex Chromosome Aneuploidy Detection in Non-Invasive Prenatal Testing.

    Directory of Open Access Journals (Sweden)

    Ting Wang

    Full Text Available Massively parallel sequencing (MPS combined with bioinformatic analysis has been widely applied to detect fetal chromosomal aneuploidies such as trisomy 21, 18, 13 and sex chromosome aneuploidies (SCAs by sequencing cell-free fetal DNA (cffDNA from maternal plasma, so-called non-invasive prenatal testing (NIPT. However, many technical challenges, such as dependency on correct fetal sex prediction, large variations of chromosome Y measurement and high sensitivity to random reads mapping, may result in higher false negative rate (FNR and false positive rate (FPR in fetal sex prediction as well as in SCAs detection. Here, we developed an optimized method to improve the accuracy of the current method by filtering out randomly mapped reads in six specific regions of the Y chromosome. The method reduces the FNR and FPR of fetal sex prediction from nearly 1% to 0.01% and 0.06%, respectively and works robustly under conditions of low fetal DNA concentration (1% in testing and simulation of 92 samples. The optimized method was further confirmed by large scale testing (1590 samples, suggesting that it is reliable and robust enough for clinical testing.

  6. Tracking fetal development through molecular analysis of maternal biofluids☆

    OpenAIRE

    Edlow, Andrea G; Bianchi, Diana W.

    2012-01-01

    Current monitoring of fetal development includes fetal ultrasonography, chorionic villus sampling or amniocentesis for chromosome analysis, and maternal serum biochemical screening for analytes associated with aneuploidy and open neural tube defects. Over the last 15 years, significant advances in noninvasive prenatal diagnosis (NIPD) via cell-free fetal (cff) nucleic acids in maternal plasma have resulted in the ability to determine fetal sex, RhD genotype, and aneuploidy. Cff nucleic acids ...

  7. Performance of Droplet Digital PCR in Non-Invasive Fetal RHD Genotyping - Comparison with a Routine Real-Time PCR Based Approach.

    Directory of Open Access Journals (Sweden)

    Iveta Svobodová

    Full Text Available Detection and characterization of circulating cell-free fetal DNA (cffDNA from maternal circulation requires an extremely sensitive and precise method due to very low cffDNA concentration. In our study, droplet digital PCR (ddPCR was implemented for fetal RHD genotyping from maternal plasma to compare this new quantification alternative with real-time PCR (qPCR as a golden standard for quantitative analysis of cffDNA. In the first stage of study, a DNA quantification standard was used. Clinical samples, including 10 non-pregnant and 35 pregnant women, were analyzed as a next step. Both methods' performance parameters-standard curve linearity, detection limit and measurement precision-were evaluated. ddPCR in comparison with qPCR has demonstrated sufficient sensitivity for analysing of cffDNA and determination of fetal RhD status from maternal circulation, results of both methods strongly correlated. Despite the more demanding workflow, ddPCR was found to be slightly more precise technology, as evaluated using quantitative standard. Regarding the clinical samples, the precision of both methods equalized with decreasing concentrations of tested DNA samples. In case of cffDNA with very low concentrations, variance parameters of both techniques were comparable. Detected levels of fetal cfDNA in maternal plasma were slightly higher than expected and correlated significantly with gestational age as measured by both methods (ddPCR r = 0.459; qPCR r = 0.438.

  8. Oxygens in DNA are main targets for ethylnitrosourea in normal and xeroderma pigmentosum fibroblasts and fetal rat brain cells

    Energy Technology Data Exchange (ETDEWEB)

    Singer, B. (Univ. of California, Berkeley); Bodell, W.J.; Cleaver, J.E.; Thomas, G.H.; Rajewsky, M.F.; Thon, W.

    1978-11-02

    One of the major problems in the study of chemical carcinogenesis is to identify in cells or animals the nucleic acid adducts known from in vitro experiments. When normal or xeroderma pigmentosum (XP) human fibroblasts or fetal rat brain cells (FBC) in culture are treated with the carcinogen, ethylnitrosourea (EtNU), to an extent permitting good cell survival (2-5 ethyl groups per 100,000 bases), it is found that the nature and proportion of the alkyl products in their DNA are remarkably similar to those found when DNA is alkylated with EtNU to a high extent in vitro (1 ethyl group per 100 bases). The products quantified in this study are ethylphosphotriesters, 7-ethylguanine (7-EtG), 3-ethyladenine (3-EtA), O/sup 6/-ethyl deoxyguanosine (O/sup 6/-EtdGuo), O/sup 2/-EtThd), 0/sup 4/-ethylthymidine and O/sup 2/-ethylcytosine (O/sup 2/-EtC). The first experimental evidence that pyrimidine oxygen atoms are major sites of reaction in cells treated with EtNU is presented. A comparison of the present model experiments with purified DNA and poly(dG) . poly(dC) with earlier studies on single-stranded polynucleotides indicates that Watson-Crick base-pairing does not significantly hinder alkylation of the oxygens. This is in contrast to previous findings concerning the very low reactivity of base-paired nitrogens. This is explained on the basis that only the oxygens have a pair of electrons not directly involved in hydrogen bonding.

  9. Environmental, Dietary, Maternal, and Fetal Predictors of Bulky DNA Adducts in Cord Blood

    DEFF Research Database (Denmark)

    Pedersen, Marie; Mendez, Michelle A; Schoket, Bernadette;

    2015-01-01

    BACKGROUND: Bulky DNA adducts reflect genotoxic exposures, have been associated with lower birth weight, and may predict cancer risk. OBJECTIVE: We selected factors known or hypothesized to affect in utero adduct formation and repair and examined their associations with adduct levels in neonates....

  10. Defective thymine dimer excision by cell-free extracts of xeroderma pigmentosum cells

    International Nuclear Information System (INIS)

    Crude extracts of normal human diploid fibroblasts and of human peripheral blood lymphocytes excise thymine dimers from purified ultraviolet-irradiated DNA, or from the DNA presumably present as chromatin in unfractionated cell-free preparations of cells that had been labeled with [3H]thymidine. Extracts of xeroderma pigmentosum cells from complementation groups A, C, and D also excise thymine dimers from purified DNA, but extracts of group A cells do not excise dimers from the DNA of radioactively labeled unfractionated cell-free preparations

  11. Integration of noninvasive prenatal prediction of fetal blood group into clinical prenatal care.

    Science.gov (United States)

    Clausen, Frederik Banch

    2014-05-01

    Incompatibility of red blood cell blood group antigens between a pregnant woman and her fetus can cause maternal immunization and, consequently, hemolytic disease of the fetus and newborn. Noninvasive prenatal testing of cell-free fetal DNA can be used to assess the risk of hemolytic disease of the fetus and newborn to fetuses of immunized women. Prediction of the fetal RhD type has been very successful and is now integrated into clinical practice to assist in the management of the pregnancies of RhD immunized women. In addition, noninvasive prediction of the fetal RhD type can be applied to guide targeted prenatal prophylaxis, thus avoiding unnecessary exposure to anti-D in pregnant women. The analytical aspect of noninvasive fetal RHD typing is very robust and accurate, and its routine utilization has demonstrated high sensitivities for fetal RHD detection. A high compliance with administering anti-D is essential for obtaining a clinical effect. Noninvasive fetal typing of RHC/c, RHE/e, and KEL may become more widely used in the future. PMID:24431264

  12. Non-invasive prenatal testing for fetal chromosome abnormalities: review of clinical and ethical issues.

    Science.gov (United States)

    Gekas, Jean; Langlois, Sylvie; Ravitsky, Vardit; Audibert, François; van den Berg, David Gradus; Haidar, Hazar; Rousseau, François

    2016-01-01

    Genomics-based non-invasive prenatal screening using cell-free DNA (cfDNA screening) was proposed to reduce the number of invasive procedures in current prenatal diagnosis for fetal aneuploidies. We review here the clinical and ethical issues of cfDNA screening. To date, it is not clear how cfDNA screening is going to impact the performances of clinical prenatal diagnosis and how it could be incorporated in real life. The direct marketing to users may have facilitated the early introduction of cfDNA screening into clinical practice despite limited evidence-based independent research data supporting this rapid shift. There is a need to address the most important ethical, legal, and social issues before its implementation in a mass setting. Its introduction might worsen current tendencies to neglect the reproductive autonomy of pregnant women. PMID:26893576

  13. Non-invasive prenatal testing for fetal chromosome abnormalities: review of clinical and ethical issues

    Science.gov (United States)

    Gekas, Jean; Langlois, Sylvie; Ravitsky, Vardit; Audibert, François; van den Berg, David Gradus; Haidar, Hazar; Rousseau, François

    2016-01-01

    Genomics-based non-invasive prenatal screening using cell-free DNA (cfDNA screening) was proposed to reduce the number of invasive procedures in current prenatal diagnosis for fetal aneuploidies. We review here the clinical and ethical issues of cfDNA screening. To date, it is not clear how cfDNA screening is going to impact the performances of clinical prenatal diagnosis and how it could be incorporated in real life. The direct marketing to users may have facilitated the early introduction of cfDNA screening into clinical practice despite limited evidence-based independent research data supporting this rapid shift. There is a need to address the most important ethical, legal, and social issues before its implementation in a mass setting. Its introduction might worsen current tendencies to neglect the reproductive autonomy of pregnant women. PMID:26893576

  14. Fetal Circulation

    Science.gov (United States)

    ... Pressure High Blood Pressure Tools & Resources Stroke More Fetal Circulation Updated:Jul 8,2016 click to enlarge The ... fetal heart. These two bypass pathways in the fetal circulation make it possible for most fetuses to survive ...

  15. DNA repair following ultraviolet and N-ethyl-N-nitrosourea treatment of cells cultured from human fetal brain, intestine, kidney, liver, and skin

    Energy Technology Data Exchange (ETDEWEB)

    Gibson-D' Ambrosio, R.E.; Leong, Y.; D' Ambrosio, S.M.

    1983-12-01

    DNA excision repair was measured in cell cultures derived from human fetal brain, intestine, kidney, liver, and skin following ultraviolet (UV) irradiation and N-ethyl-N-nitrosourea (ENU) treatment. Cells in early passages were exposed to 5 or 10 J of UV radiation per sq m or to 25 microM to 3.5 mM ENU. DNA excision repair was determined by (a) scintillation counting and autoradiography to measure unscheduled DNA synthesis (UDS) and (b) the UV-endonuclease-sensitive site assay to measure pyrimidine dimers directly. The level of UDS following treatment of these cell cultures with UV was both time and dose dependent. UDS also increased with increasing doses of ENU up to 350 microM but decreased at doses greater than 500 microM. Cells derived from human fetal brain, kidney, and liver appeared to exhibit lower (50 to 80%) levels of UDS following UV irradiation or ENU treatment than did cells cultured from human fetal skin or intestine. The loss of UV-endonuclease-sensitive sites assayed in skin, liver, and kidney cells over a 24-hr period confirmed the differences observed by UDS in these cells. Skin cells removed 50% of the initial pyrimidine dimers from their DNA within an 8-hr period and 65 to 86% in 24 hr. Kidney and liver cells, on the other hand, removed only 28 and 32% of the initial dimers, respectively, over a 24-hr period. The data suggest differential excision repair responses following UV irradiation and ENU treatment of cells derived from different human fetal organs.

  16. Adverse effects of trichothiodystrophy DNA repair and transcription gene disorder on human fetal development

    OpenAIRE

    Moslehi, R; Signore, C; Tamura, D; Mills, JL; DiGiovanna, JJ; Tucker, MA; Troendle, J; Ueda, T; Boyle, J.; Khan, SG; Oh, K-S; Goldstein, AM; Kraemer, KH

    2009-01-01

    The effects of DNA repair and transcription gene abnormalities in human pre-natal life have never been studied. Trichothiodystrophy (TTD) is a rare (affected frequency of 10−6) recessive disorder caused by mutations in genes involved in nucleotide excision repair (NER) pathway and in transcription. Based on our novel clinical observations, we conducted a genetic epidemiologic study to investigate gestational outcomes associated with TTD. We compared pregnancies resulting in TTD-affected offsp...

  17. GST-π EXPRESSION IN TRANSFORMED CELLS BY TRANSFECTING OF DNA ISOLATED FROM HUMAN FETAL LUNG TISSUES TREATED WITH CARCINOGENS

    Institute of Scientific and Technical Information of China (English)

    Yao Denggao; Hu Guogang; Luo Xianmao; Zhu Ming

    1998-01-01

    Objective: To investigate the relationship between the GSTs, GST-π expression and initiation of lung carcinogenesis. Methods: The Rat-1 cells were transformed by carcinogens (DEN, MNU and CSC) treated fetal lung DNA for 24 h. Results: The GSTs activities toward 1-chloro-2, 4-dinitro-benzene (CDNB) in transformed cells were significantly higher than in the solvent control cells (P<0.05). GST-π content and GST-π mRNA expression level of transformed cells were also higher than those of control cells which were performed by ELISA and Northern blotting method respectively. The results indicated that the higher GSTS activities of transformed cells were due to the increase of GST-π content and the GST-π mRNA overexpressing may be responsible for the increase of GST-π protein level of the transformed cells. Conclusion: The changes of GSTs and GST-π may be considered as the one of the biomarkers of the initiation of human lung carcinogenesis.

  18. cDNA Cloning Demonstrates the Expression of Pregnancy-Specific Glycoprotein Genes, a Subgroup of the Carcinoembryonic Antigen Gene Family, in Fetal Liver

    OpenAIRE

    Zimmermann, Wolfgang; Weiss, Martina; Thompson, John A.

    1989-01-01

    The pregnancy-specific glycoprotein (PSG) genes constitute a subgroup of the carcinoembryonic antigen (CEA) gene family. Here we report the cloning of four cDNAs coding for different members of the PSG family from a human fetal liver cDNA library. They are derived from three closely related genes (PSG1, PSG4 and PSG6). Two of the cDNA clones represent splice variants of PSG1 (PSG1a, PSG1d) differing in their C-terminal domain and 3′-untranslated regions. All encoded proteins show the same dom...

  19. Adverse effects of trichothiodystrophy DNA repair and transcription gene disorder on human fetal development.

    Science.gov (United States)

    Moslehi, R; Signore, C; Tamura, D; Mills, J L; Digiovanna, J J; Tucker, M A; Troendle, J; Ueda, T; Boyle, J; Khan, S G; Oh, K-S; Goldstein, A M; Kraemer, K H

    2010-04-01

    The effects of DNA repair and transcription gene abnormalities in human pre-natal life have never been studied. Trichothiodystrophy (TTD) is a rare (affected frequency of 10(-6)) recessive disorder caused by mutations in genes involved in nucleotide excision repair (NER) pathway and in transcription. Based on our novel clinical observations, we conducted a genetic epidemiologic study to investigate gestational outcomes associated with TTD. We compared pregnancies resulting in TTD-affected offspring (n = 24) with respect to abnormalities during their antenatal and neonatal periods to pregnancies resulting in their unaffected siblings (n = 18), accounting for correlation, and to population reference values. Significantly higher incidence of several severe gestational complications was noted in TTD-affected pregnancies. Small for gestational age (SGA) maternal serum human chorionic gonadotropin (hCG) levels (RR = 14.3, 95% CI = 7.0-16.6, p constellation of observed complications in our study. Thus, we hypothesize that TTD DNA repair and transcription genes play an important role in normal human placental development. PMID:20002457

  20. Mechanism of estrogen receptor-dependent transcription in a cell-free system.

    OpenAIRE

    Elliston, J F; Fawell, S E; Klein-Hitpass, L; Tsai, S. Y.; Tsai, M J; Parker, M G; O'Malley, B W

    1990-01-01

    RNA synthesis was stimulated directly in a cell-free expression system by crude preparations of recombinant mouse estrogen receptor (ER). Receptor-stimulated transcription required the presence of estrogen response elements (EREs) in the test template and could be specifically inhibited by addition of competitor oligonucleotides containing EREs. Moreover, polyclonal antibodies directed against the DNA-binding region of ER inhibited ER-dependent transcription. In our cell-free expression syste...

  1. Noninvasive prenatal testing by maternal plasma DNA analysis: current practice and future applications.

    Science.gov (United States)

    Chiu, Rossa W K

    2014-01-01

    Prenatal screening of fetal chromosomal aneuploidies and some common genetic diseases is an integral part of antenatal care. Definitive prenatal diagnosis is conventionally achieved by the sampling of fetal genetic material by amniocentesis or chorionic villus sampling. Due to the invasiveness of those procedures, they are associated with a 1 in 200 chance of fetal miscarriage. Hence, researchers have been exploring noninvasive ways to sample fetal genetic material. The presence of cell-free DNA released by the fetus into the circulation of its mother was demonstrated in 1997. Circulating fetal DNA is therefore obtainable through the collection of a blood sample from the pregnant woman without posing any physical harm to the fetus. By analyzing this source of fetal genetic material, researchers have succeeded in developing DNA-based noninvasive tests for the assessment of Down syndrome and single gene diseases. Since the end of 2011, tests for the noninvasive assessment of chromosomal aneuploidies have become commercially available in parts of the world. Recommendations from professional groups have since been made regarding how these tests could be incorporated into the framework of existing prenatal screening programs. More recently, cell-free circulating fetal DNA analysis have been shown to be applicable to the deciphering of the fetal molecular karyotype, genome and methylome. It is envisioned that an increasing number of the noninvasive prenatal tests will become clinically available. The ethical, social and legal implications of the introduction of some of these tests would need to be discussed in the context of different cultures, societal values and the legal framework. PMID:25083893

  2. High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients

    OpenAIRE

    Kukita, Yoji; Matoba, Ryo; Uchida, Junji; Hamakawa, Takuya; Doki, Yuichiro; Imamura, Fumio; Kato, Kikuya

    2015-01-01

    Circulating tumour DNA (ctDNA) is an emerging field of cancer research. However, current ctDNA analysis is usually restricted to one or a few mutation sites due to technical limitations. In the case of massively parallel DNA sequencers, the number of false positives caused by a high read error rate is a major problem. In addition, the final sequence reads do not represent the original DNA population due to the global amplification step during the template preparation. We established a high-fi...

  3. Trinucleotide repeat expansions catalyzed by human cell-free extracts

    Institute of Scientific and Technical Information of China (English)

    Jennifer R Stevens; Elaine E Lahue; Guo-Min Li; Robert S Lahue

    2013-01-01

    Trinucleotide repeat expansions cause 17 heritable human neurological disorders.In some diseases,somatic expansions occur in non-proliferating tissues such as brain where DNA replication is limited.This finding stimulated significant interest in replication-independent expansion mechanisms.Aberrant DNA repair is a likely source,based in part on mouse studies showing that somatic expansions are provoked by the DNA repair protein MutSβ (Msh2-Msh3complex).Biochemical studies to date used cell-free extracts or purified DNA repair proteins to yield partial reactions at triplet repeats.The findings included expansions on one strand but not the other,or processing of DNA hairpin structures thought to be important intermediates in the expansion process.However,it has been difficult to recapitulate complete expansions in vitro,and the biochemical role of MutSβ remains controversial.Here,we use a novel in vitro assay to show that human cell-free extracts catalyze expansions and contractions of trinucleotide repeats without the requirement for DNA replication.The extract promotes a size range of expansions that is similar to certain diseases,and triplet repeat length and sequence govern expansions in vitro as in vivo.MutSβ stimulates expansions in the extract,consistent with aberrant repair of endogenous DNA damage as a source of expansions.Overall,this biochemical system retains the key characteristics of somatic expansions in humans and mice,suggesting that this important mutagenic process can be restored in the test tube.

  4. Fetal echocardiography

    International Nuclear Information System (INIS)

    USG performed with a high-end machine, using a good cine-loop facility is extremely helpful in the diagnosis of fetal cardiac anomalies. In fetal echocardiography, the four-chamber view and the outflow-tract view are used to diagnose cardiac anomalies. The most important objective during a targeted anomaly scan is to identify those cases that need a dedicated fetal echocardiogram. Associated truncal and chromosomal anomalies need to be identified. This review shows how fetal echocardiography, apart from identifying structural defects in the fetal heart, can be used to look at rhythm abnormalities and other functional aspects of the fetal heart

  5. In-cell PCR method for specific genotyping of genomic DNA from one individual in a mixture of cells from two individuals: a model study with specific relevance to prenatal diagnosis based on fetal cells in maternal blood

    DEFF Research Database (Denmark)

    Hviid, T Vauvert

    2002-01-01

    BACKGROUND: During recent years, much attention has been paid to the possibility of using fetal cells circulating in the pregnant woman's blood for prenatal diagnosis of genetic or chromosomal abnormalities. Although successes have been achieved in enrichment procedures for fetal cells from...... sequence only in the male cells, leading to the correct HLA-DPB1 genotyping of the male by DNA sequencing of a nested, linked TSPY-HLA-DPB1 PCR product. CONCLUSION: This approach might be usable on mixed cell populations of fetal and maternal cells obtained after conventional cell-sorting techniques on...

  6. Fetal Heart

    Science.gov (United States)

    ... the right atrium to the left atrium during fetal development, but closes after birth. So the ductus arteriosus and the foramen ovale are part of the fetal circulatory system before birth but disappear soon after ...

  7. Fetal development

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/002398.htm Fetal development To use the sharing features on this page, ... Cunningham FG, Leveno KJ, Bloom SL, et al. Fetal growth and development. In: Cunningham FG, Leveno KL, Bloom SL, et ...

  8. Correction: Grömminger, S., et al. Fetal Aneuploidy Detection by Cell-Free DNA Sequencing for Multiple Pregnancies and Quality Issues with Vanishing Twins. J. Clin. Med. 2014, 3, 679-692

    Directory of Open Access Journals (Sweden)

    Sebastian Grömminger

    2014-11-01

    Full Text Available The authors wish to make the following corrections to this paper [1]: On page 683 at the end of Section 3.2. lines 13–14, the word “no” is missing. The correct sentence should be: “There has been no evidence of false-negative NIPT results so far in the pregnancies included in this study.”[...

  9. Non-invasive prenatal testing for fetal chromosome abnormalities: review of clinical and ethical issues

    Directory of Open Access Journals (Sweden)

    Gekas J

    2016-02-01

    Full Text Available Jean Gekas,1,2 Sylvie Langlois,3 Vardit Ravitsky,4 François Audibert,5 David Gradus van den Berg,6 Hazar Haidar,4 François Rousseau2,7 1Prenatal Diagnosis Unit, Department of Medical Genetics and Pediatrics, Faculty of Medicine, Université Laval, Québec City, QC, Canada; 2Department of Medical Biology, CHU de Québec, Québec City, QC, Canada; 3Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada; 4Bioethics Program, Department of Social and Preventive Medicine, School of Public Health, University of Montreal, Montreal, QC, Canada; 5Department of Obstetrics and Gynecology, Hospital Sainte-Justine, Montreal, QC, Canada; 6Department of Social and Preventive Medicine, Faculty of Medicine, Université Laval, Québec City, QC, Canada; 7Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, Université Laval, Québec City, QC, Canada Abstract: Genomics-based non-invasive prenatal screening using cell-free DNA (cfDNA screening was proposed to reduce the number of invasive procedures in current prenatal diagnosis for fetal aneuploidies. We review here the clinical and ethical issues of cfDNA screening. To date, it is not clear how cfDNA screening is going to impact the performances of clinical prenatal diagnosis and how it could be incorporated in real life. The direct marketing to users may have facilitated the early introduction of cfDNA screening into clinical practice despite limited evidence-based independent research data supporting this rapid shift. There is a need to address the most important ethical, legal, and social issues before its implementation in a mass setting. Its introduction might worsen current tendencies to neglect the reproductive autonomy of pregnant women. Keywords: prenatal diagnosis, Down syndrome, non-invasive prenatal testing, cell-free fetal DNA, informed consent, reproductive autonomy

  10. DNA binding mode of the cis and trans geometries of new antitumor nonclassical platinum complexes containing piperidine, piperazine or 4-picoline ligand in cell-free media. Relations to their activity in cancer cell lines

    Czech Academy of Sciences Publication Activity Database

    Kašpárková, Jana; Marini, Victoria; Najajreh, Y.; Gibson, D.; Brabec, Viktor

    2003-01-01

    Roč. 42, č. 20 (2003), s. 6321-6332. ISSN 0006-2960 R&D Projects: GA AV ČR IAA5004101; GA AV ČR KJB5004301; GA ČR GA305/01/0418 Institutional research plan: CEZ:AV0Z5004920 Keywords : cross links * DNA * nonclassical platinum complexes Subject RIV: BO - Biophysics Impact factor: 3.922, year: 2003

  11. DNA and glutathione interactions in cell-free media of asymmetric platinum(II) complexes cis- and trans-[PtCl2(isopropylamine)(1-methylimidazole)]: relations to their different antitumor effects

    Czech Academy of Sciences Publication Activity Database

    Suchánková, Tereza; Vojtíšková, Marie; Reedijk, J.; Brabec, Viktor; Kašpárková, Jana

    2009-01-01

    Roč. 14, č. 1 (2009), s. 75-87. ISSN 0949-8257 R&D Projects: GA MŠk(CZ) LC06030; GA MŠk(CZ) ME08017; GA MŠk(CZ) OC08003; GA AV ČR(CZ) 1QS500040581; GA AV ČR(CZ) KAN200200651; GA AV ČR(CZ) IAA400040803; GA MZd(CZ) NR8562 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : DNA * platinum * cancer Subject RIV: BO - Biophysics Impact factor: 3.415, year: 2009

  12. Quantification of Cell-Free mSHOX2 Plasma DNA for Therapy Monitoring in Advanced Stage Non-Small Cell (NSCLC) and Small-Cell Lung Cancer (SCLC) Patients

    OpenAIRE

    Schmidt, Bernd; Beyer, Julia; Dietrich, Dimo; Bork, Ines; Liebenberg, Volker; Fleischhacker, Michael

    2015-01-01

    Purpose Most patients suffering from advanced lung cancer die within a few months. To exploit new therapy regimens we need better methods for the assessment of a therapy response. Material and Methods In a pilot study we prospectively enrolled 36 patients with advanced NSCLC and SCLC (34 stage IV, 2 stage IIIB) of whom 34 received standard platinum-based chemo/radiotherapy and two were treated with a tyrosine kinase inhibitor. We measured the levels of extracellular methylated SHOX2 DNA (mSHO...

  13. Quantification of cell-free mSHOX2 Plasma DNA for therapy monitoring in advanced stage non-small cell (NSCLC and small-cell lung cancer (SCLC patients.

    Directory of Open Access Journals (Sweden)

    Bernd Schmidt

    Full Text Available Most patients suffering from advanced lung cancer die within a few months. To exploit new therapy regimens we need better methods for the assessment of a therapy response.In a pilot study we prospectively enrolled 36 patients with advanced NSCLC and SCLC (34 stage IV, 2 stage IIIB of whom 34 received standard platinum-based chemo/radiotherapy and two were treated with a tyrosine kinase inhibitor. We measured the levels of extracellular methylated SHOX2 DNA (mSHOX2 in plasma before and during therapy until re-staging. The mSHOX2 analysis was blinded with respect to the clinical data making it an observational study.According to the re-staging of 31 first-line patients, 19 patients were classified as non-responders while 12 patients were in the responder group. We observed a tight correlation between radiological data and the change of plasma mSHOX2 level as the equivalent for a therapy response. A ROC analysis showed a high discriminatory power for both patient groups already one week after therapy start (AUC 0.844. Additionally, a Kaplan-Meier and Cox Proportional Hazards analyses revealed a strong relationship between survival and plasma mSHOX2 value p ≤ 0.001 (hazard ratio 11.08 providing some evidence for mSHOX2 also being a predictive marker.The longitudinal measurement of extracellular plasma mSHOX2 DNA yields information about the response to cytotoxic treatment and allows an early assessment of treatment response for lung cancer patients. If confirmed in a larger study this would be a valuable tool for selecting and guiding a cytotoxic treatment.

  14. Quantification of Cell-Free mSHOX2 Plasma DNA for Therapy Monitoring in Advanced Stage Non-Small Cell (NSCLC) and Small-Cell Lung Cancer (SCLC) Patients

    Science.gov (United States)

    Schmidt, Bernd; Beyer, Julia; Dietrich, Dimo; Bork, Ines; Liebenberg, Volker; Fleischhacker, Michael

    2015-01-01

    Purpose Most patients suffering from advanced lung cancer die within a few months. To exploit new therapy regimens we need better methods for the assessment of a therapy response. Material and Methods In a pilot study we prospectively enrolled 36 patients with advanced NSCLC and SCLC (34 stage IV, 2 stage IIIB) of whom 34 received standard platinum-based chemo/radiotherapy and two were treated with a tyrosine kinase inhibitor. We measured the levels of extracellular methylated SHOX2 DNA (mSHOX2) in plasma before and during therapy until re-staging. The mSHOX2 analysis was blinded with respect to the clinical data making it an observational study. Results According to the re-staging of 31 first-line patients, 19 patients were classified as non-responders while 12 patients were in the responder group. We observed a tight correlation between radiological data and the change of plasma mSHOX2 level as the equivalent for a therapy response. A ROC analysis showed a high discriminatory power for both patient groups already one week after therapy start (AUC 0.844). Additionally, a Kaplan-Meier and Cox Proportional Hazards analyses revealed a strong relationship between survival and plasma mSHOX2 value p≤0.001 (hazard ratio 11.08) providing some evidence for mSHOX2 also being a predictive marker. Conclusion The longitudinal measurement of extracellular plasma mSHOX2 DNA yields information about the response to cytotoxic treatment and allows an early assessment of treatment response for lung cancer patients. If confirmed in a larger study this would be a valuable tool for selecting and guiding a cytotoxic treatment. PMID:25675432

  15. Non invasive prenatal diagnosis: analysis of circulating fetal DNA and cells in maternal blood El diagnóstico prenatal no invasor: análisis de células y ADN fetal circulantes en la sangre materna

    Directory of Open Access Journals (Sweden)

    Diana Cecilia Jaramillo Posada

    2009-11-01

    Full Text Available

    Prenatal non invasive diagnosis by means of analyses of foetal DNA or cells circulating in maternal blood is one of the most promising areas of obstetrics. Among maternal diseases that could be diagnosed by these methods, or whose behaviour could be predicted, are preeclampsia, growth restriction and preterm labour. Some foetal conditions that could be detected are sex, chromosomal anomalies and single-gene defects. However, these are complex and expensive techniques that are not regularly performed in health care institutions. With this review we intend to provide the readers with up to date information on the main techniques available for the study of circulating foetal cells and DNA, and on their possible clinical applications. The review was based on a search for journals indexed up to 2008 in Pubmed, Scielo and Latindex. Especially relevant articles were chosen by the authors.

    El diagnóstico prenatal temprano y no invasor por medio del análisis de células o ADN fetales circulantes en la sangre materna es un área prometedora de la obstetricia moderna. Entre las enfermedades que se pueden diagnosticar o cuyo comportamiento es posible predecir por estos métodos se encuentran la preeclampsia, la restricción del crecimiento intrauterino y el parto pretérmino. Algunas condiciones fetales que podrían detectarse son el sexo, ciertas anomalías cromosómicas y los defectos de un solo gen. Sin

  16. Inhibitory effect of benzene metabolites on nuclear DNA synthesis in bone marrow cells

    International Nuclear Information System (INIS)

    Effects of endogenously produced and exogenously added benzene metabolites on the nuclear DNA synthetic activity were investigated using a culture system of mouse bone marrow cells. Effects of the metabolites were evaluated by a 30-min incorporation of [3H]thymidine into DNA following a 30-min interaction with the cells in McCoy's 5a medium with 10% fetal calf serum. Phenol and muconic acid did not inhibit nuclear DNA synthesis. However, catechol, 1,2,4-benzenetriol, hydroquinone, and p-benzoquinone were able to inhibit 52, 64, 79, and 98% of the nuclear DNA synthetic activity, respectively, at 24 μM. In a cell-free DNA synthetic system, catechol and hydroquinone did not inhibit the incorporation of [3H]thymidine triphosphate into DNA up to 24 μM but 1,2,4-benzenetriol and p-benzoquinone did. The effect of the latter two benzene metabolites was completely blocked in the presence of 1,4-dithiothreitol (1 mM) in the cell-free assay system. Furthermore, when DNA polymerase α, which requires a sulfhydryl (SH) group as an active site, was replaced by DNA polymerase 1, which does not require an SH group for its catalytic activity, p-benzoquinone and 1,2,4-benzenetriol were unable to inhibit DNA synthesis. Thus, the data imply the p-benzoquinone and 1,2,4-benzenetriol inhibited DNA polymerase α, consequently resulting in inhibition of DNA synthesis in both cellular and cell-free DNA synthetic systems. The present study identifies catechol, hydroquinone, p-benzoquinone, and 1,2,4-benzenetriol as toxic benzene metabolites in bone marrow cells and also suggests that their inhibitory action on DNA synthesis is mediated by mechanism(s) other than that involving DNA damage as a primary cause

  17. Fetal echocardiography

    Science.gov (United States)

    ... Hori D. Fetal arrhythmia: prenatal diagnosis and perinatal management. J Obstet Gynaecol Res . 2009 ... G Lee, MD, MSc, IBCLC, Associate Professor of Pediatrics, Division of Neonatology, Medical University of ...

  18. Single-Stranded DNA-Binding Transcriptional Regulator FUBP1 Is Essential for Fetal and Adult Hematopoietic Stem Cell Self-Renewal

    Directory of Open Access Journals (Sweden)

    Uta Rabenhorst

    2015-06-01

    Full Text Available The ability of hematopoietic stem cells (HSCs to self-renew is a prerequisite for the establishment of definitive hematopoiesis and life-long blood regeneration. Here, we report the single-stranded DNA-binding transcriptional regulator far upstream element (FUSE-binding protein 1 (FUBP1 as an essential factor of HSC self-renewal. Functional inactivation of FUBP1 in two different mouse models resulted in embryonic lethal anemia at around E15.5 caused by severely diminished HSCs. Fetal and adult HSCs lacking FUBP1 revealed an HSC-intrinsic defect in their maintenance, expansion, and long-term blood reconstitution, but could differentiate into all hematopoietic lineages. FUBP1-deficient adult HSCs exhibit significant transcriptional changes, including upregulation of the cell-cycle inhibitor p21 and the pro-apoptotic Noxa molecule. These changes caused an increase in generation time and death of HSCs as determined by video-microscopy-based tracking. Our data establish FUBP1 and its recognition of single-stranded genomic DNA as an important element in the transcriptional regulation of HSC self-renewal.

  19. Single-Stranded DNA-Binding Transcriptional Regulator FUBP1 Is Essential for Fetal and Adult Hematopoietic Stem Cell Self-Renewal.

    Science.gov (United States)

    Rabenhorst, Uta; Thalheimer, Frederic B; Gerlach, Katharina; Kijonka, Marek; Böhm, Stefanie; Krause, Daniela S; Vauti, Franz; Arnold, Hans-Henning; Schroeder, Timm; Schnütgen, Frank; von Melchner, Harald; Rieger, Michael A; Zörnig, Martin

    2015-06-30

    The ability of hematopoietic stem cells (HSCs) to self-renew is a prerequisite for the establishment of definitive hematopoiesis and life-long blood regeneration. Here, we report the single-stranded DNA-binding transcriptional regulator far upstream element (FUSE)-binding protein 1 (FUBP1) as an essential factor of HSC self-renewal. Functional inactivation of FUBP1 in two different mouse models resulted in embryonic lethal anemia at around E15.5 caused by severely diminished HSCs. Fetal and adult HSCs lacking FUBP1 revealed an HSC-intrinsic defect in their maintenance, expansion, and long-term blood reconstitution, but could differentiate into all hematopoietic lineages. FUBP1-deficient adult HSCs exhibit significant transcriptional changes, including upregulation of the cell-cycle inhibitor p21 and the pro-apoptotic Noxa molecule. These changes caused an increase in generation time and death of HSCs as determined by video-microscopy-based tracking. Our data establish FUBP1 and its recognition of single-stranded genomic DNA as an important element in the transcriptional regulation of HSC self-renewal. PMID:26095368

  20. Alteration of Gene Expression, DNA Methylation, and Histone Methylation in Free Radical Scavenging Networks in Adult Mouse Hippocampus following Fetal Alcohol Exposure

    Science.gov (United States)

    Chater-Diehl, Eric J.; Castellani, Christina A.; Alberry, Bonnie L.; Singh, Shiva M.

    2016-01-01

    The molecular basis of Fetal Alcohol Spectrum Disorders (FASD) is poorly understood; however, epigenetic and gene expression changes have been implicated. We have developed a mouse model of FASD characterized by learning and memory impairment and persistent gene expression changes. Epigenetic marks may maintain expression changes over a mouse’s lifetime, an area few have explored. Here, mice were injected with saline or ethanol on postnatal days four and seven. At 70 days of age gene expression microarray, methylated DNA immunoprecipitation microarray, H3K4me3 and H3K27me3 chromatin immunoprecipitation microarray were performed. Following extensive pathway analysis of the affected genes, we identified the top affected gene expression pathway as “Free radical scavenging”. We confirmed six of these changes by droplet digital PCR including the caspase Casp3 and Wnt transcription factor Tcf7l2. The top pathway for all methylation-affected genes was “Peroxisome biogenesis”; we confirmed differential DNA methylation in the Acca1 thiolase promoter. Altered methylation and gene expression in oxidative stress pathways in the adult hippocampus suggests a novel interface between epigenetic and oxidative stress mechanisms in FASD. PMID:27136348

  1. Molecular cloning and in silico analysis of the duck (Anas platyrhynchos MEF2A gene cDNA and its expression profile in muscle tissues during fetal development

    Directory of Open Access Journals (Sweden)

    Hehe Liu

    2012-01-01

    Full Text Available The role of myogenic enhancer transcription factor 2a (MEF2A in avian muscle during fetal development is unknown. In this work, we cloned the duck MEF2A cDNA sequence (GenBank accession no. HM460752 and examined its developmental expression profiles in cardiac muscle, non-vascular smooth muscle and skeletal muscle. Duck MEF2A cDNA comprised 1479 bp encoding 492 amino acid residues. In silico analysis showed that MEF2A contained MADS (MCM1, AGAMOUS, DEFICIENS and SRF -serum response factor, MEF2 and mitogen-activated protein kinase (MAPK transcription domains with high homology to related proteins in other species. Modified sites in these domains were conserved among species and several variants were found. Quantitative PCR showed that MEF2A was expressed in all three muscles at each developmental stage examined, with the expression in smooth muscle being higher than in the other muscles. These results indicate that the conserved domains of duck MEF2A, including the MADS and MEF2 domains, are important for MEF2A transcription factor function. The expression of MEF2A in duck smooth muscle and cardiac muscle suggests that MEF2A plays a role in these two tissues.

  2. Release of cell-free ice nuclei by Erwinia herbicola.

    OpenAIRE

    Phelps, P; Giddings, T. H.; Prochoda, M; Fall, R

    1986-01-01

    Several ice-nucleating bacterial strains, including Erwinia herbicola, Pseudomonas fluorescens, and Pseudomonas syringae isolates, were examined for their ability to shed ice nuclei into the growth medium. Only E. herbicola isolates shed cell-free ice nuclei active at -2 to -10 degrees C. These cell-free nuclei exhibited a freezing spectrum similar to that of ice nuclei found on whole cells, both above and below -5 degrees C. Partially purified cell-free nuclei were examined by density gradie...

  3. Cell-free circulating tumor DNA in cancer

    OpenAIRE

    Qin, Zhen; Ljubimov, Vladimir A.; Zhou, Cuiqi; Tong, Yunguang; Liang, Jimin

    2016-01-01

    Cancer is a common cause of death worldwide. Despite significant advances in cancer treatments, the morbidity and mortality are still enormous. Tumor heterogeneity, especially intratumoral heterogeneity, is a significant reason underlying difficulties in tumor treatment and failure of a number of current therapeutic modalities, even of molecularly targeted therapies. The development of a virtually noninvasive “liquid biopsy” from the blood has been attempted to characterize tumor heterogeneit...

  4. Plasma cell-free DNA in diagnostic KRAS mutation testing

    OpenAIRE

    Isomursu, Aleksi

    2015-01-01

    Epidermaalisen kasvutekijän reseptorin (EGFR) monoklonaalisia vasta-aineita (mAb) käytetään apuna metastaattisen kolorektaalisyövän (mCRC) hoidossa. Aktivoivat mutaatiot KRAS- ja NRAS-onkogeeneissä tekevät syöpäsolut vastustuskykyisiksi anti-EGFR-vasta-aineille, ja tästä syystä tuumorit tulee genotyypittää ennen hoidon aloittamista. Kudosbiopsian ottaminen on kuitenkin invasiivinen toimenpide, eikä näyte välttämättä edusta kattavasti syövän kaikkien solupopulaatioiden geneettisiä muutoksia. V...

  5. Fetal cardiology

    International Nuclear Information System (INIS)

    Doppler echocardiography makes it possible to diagnose congenital heart disease in early pregnancy. It allows us to study the anatomical configuration of the fetal heart, and additionally, to evaluate the physiological conditions of the fetus. Evaluation of the direction, velocity, wave form pattern, and quantification of blood flow at the various sites in the fetal heart helps us to assess the characteristics of the fetal circulation and condition of the fetal heart. In order to use this technique in pathological situations, an initial study of the developing normal human fetal circulation was necessary. The authors studied 34 uncomplicated pregnancies by serial Doppler echocardiography. The studies were performed every 4 weeks from 16-weeks gestation to term. The pulsed Doppler sector scanner provided cardiac cross-sectional images, mitral and tricuspid blood velocities were obtained from apical four-chamber views. Angle corrected maximal and mean temporal velocities were calculated by digitizing the Doppler frequency shift recording on a graphic tablet computed with a minicomputer. The angle between the Doppler interrogation beam and the direction of blood flow was kept as small as possible in order to minimize the error

  6. Fetal MRI

    International Nuclear Information System (INIS)

    New, ultrafast sequences have made it possible to obtain MR images of the fetus without maternal sedation or immobilization of the fetus itself. While fetal MRI began as an adjunct to ultrasound, it has now been shown that MRI can provide additional information that may change prognosis, the management of pregnancy, or the treatment of the newborn child. It is of particular value in the assessment of malformations of the central nervous system. The steady development and adaptation of MR-sequences to the needs of fetal imaging has led to new indications that can support prognostic and therapeutic decisions. (orig.)

  7. Fetal MRI

    Energy Technology Data Exchange (ETDEWEB)

    Prayer, D.; Brugger, P.C. [University Hospital of Vienna (Austria). Division of Neuroradiology

    2004-07-01

    New, ultrafast sequences have made it possible to obtain MR images of the fetus without maternal sedation or immobilization of the fetus itself. While fetal MRI began as an adjunct to ultrasound, it has now been shown that MRI can provide additional information that may change prognosis, the management of pregnancy, or the treatment of the newborn child. It is of particular value in the assessment of malformations of the central nervous system. The steady development and adaptation of MR-sequences to the needs of fetal imaging has led to new indications that can support prognostic and therapeutic decisions. (orig.)

  8. Fetal MRI

    International Nuclear Information System (INIS)

    Ultrasonography is the method of choice for prenatal malformation screening, but it does not always provide sufficient information for correct diagnosis or adequate abnormality evaluation. Fetal MRI is increasingly being used to complete sonographic findings. It was initially used for evaluation of cerebral abnormalities but is increasingly being applied to other fetal areas. In vivo investigation of fetal brain maturation has been enhanced by MRI. An adequate analysis of fetal chest and abdomen can be achieved with fast T2-, T1-weighted and diffusion-weighted imaging (DWI). The advantages include the great field of view and the excellent soft tissue contrast. This allows correct diagnosis of congenital diaphragmatic hernia and evaluation of the consequences on pulmonary growth. Other pulmonary malformations, such as cystic adenomatoid malformation, sequestration and brochogenic cysts, can also be easily identified. Renal position can be quickly determined using DWI sequences and renal agenesia can be easily diagnosed with only one sequence. Prenatal MRI is virtually as effective as postnatal examination, dispenses with transport of a potentially very ill newborn, and provides logistic advantages. Therefore, prenatal MRI is useful for adequate postnatal treatment of newborns with malformations. (orig.)

  9. Ion Torrent PGMTM系统检测孕妇血浆中胎儿游离DNA%Application of Ion Torrent PGMTM System in Detection of Fetal DNA in Maternal Plasma

    Institute of Scientific and Technical Information of China (English)

    刘亚楠; 赵雪莹; 平原; 徐庆文; 黄江平; 邹凯南; 周怀谷

    2015-01-01

    目的:探讨Ion Torrent PGMTM系统用于孕妇外周血血浆中胎儿游离DNA的Y-STR检测的可行性。方法采集8例孕晚期(38周)和8例孕中期(12周)的无关孕妇外周血血浆,提取胎儿游离DNA,对DYS390、DYS391、DYS393、DYS438、DYS437、DYS456、DYS635共7个基因座进行复合扩增,在Ion Torrent PGMTM系统进行Y-STR测序分析和毛细管电泳分析,并将两者结果进行比较。结果在分娩男性胎儿的孕晚期及中期孕妇血浆中均检测到Y-STR片段,测序检测得出的基因座比毛细管电泳检测得出的多,且与分娩胎儿的Y-STR基因型相一致。结论 Ion Torrent PGMTM系统用于孕妇外周血血浆胎儿游离DNA的Y-STR检测,具有高灵敏度、高通量的特点,且有较好的法医学应用价值。%Objective To explore the feasibility of detecting of Y-STR of fetal DNA in m aternal plasm a using Ion Torrent PGMTM System . Methods A total of 16 fetal DNA sam ples from m aternal plasm as (8 cases from 38 w eeks gestational age and 8 ones from 12 w eeks) w ere prepared and a m ultiplex assay w ith 7 STR loci (DYS390,DYS391,DYS393,DYS438,DYS437,DYS456,DYS635) w as designed for m ul-tiplex-PC R am plification. U sing Ion Torrent PGMTM System , the results of Y-STR sequences and capillary electrophoresis w ere obtained and com pared. Results Y-STR specific alleles w ere detected in the m ater-nal plasm a of all the pregnant w om en having m ale babies of second and third trim ester, w hich w ere higher than that detected by capillary electrophoresis. C onsistent Y-STR genotypes w ere observed betw een fetal DNA from m aternal plasm a and genom ic DNA from the new born babies. Conclusion B ased on Ion Torrent PGMTM System , the prenatal Y-STR detection m ethod m ay provide a high-sensitive and high-throughput choice for prenatal STR detection in forensic testing.

  10. Comparative analysis of eukaryotic cell-free expression systems.

    Science.gov (United States)

    Hartsough, Emily M; Shah, Pankti; Larsen, Andrew C; Chaput, John C

    2015-09-01

    Cell-free protein synthesis (CFPS) allows researchers to rapidly generate functional proteins independent of cell culture. Although advances in eukaryotic lysates have increased the amount of protein that can be produced, the nuances of different translation systems lead to variability in protein production. To help overcome this problem, we have compared the relative yield and template requirements for three commonly used commercial cell-free translation systems: wheat germ extract (WGE), rabbit reticulocyte lysate (RRL), and HeLa cell lysate (HCL). Our results provide a general guide for researchers interested in using cell-free translation to generate recombinant protein for biomedical applications. PMID:26345507

  11. Methylomic trajectories across human fetal brain development.

    Science.gov (United States)

    Spiers, Helen; Hannon, Eilis; Schalkwyk, Leonard C; Smith, Rebecca; Wong, Chloe C Y; O'Donovan, Michael C; Bray, Nicholas J; Mill, Jonathan

    2015-03-01

    Epigenetic processes play a key role in orchestrating transcriptional regulation during development. The importance of DNA methylation in fetal brain development is highlighted by the dynamic expression of de novo DNA methyltransferases during the perinatal period and neurodevelopmental deficits associated with mutations in the methyl-CpG binding protein 2 (MECP2) gene. However, our knowledge about the temporal changes to the epigenome during fetal brain development has, to date, been limited. We quantified genome-wide patterns of DNA methylation at ∼ 400,000 sites in 179 human fetal brain samples (100 male, 79 female) spanning 23 to 184 d post-conception. We identified highly significant changes in DNA methylation across fetal brain development at >7% of sites, with an enrichment of loci becoming hypomethylated with fetal age. Sites associated with developmental changes in DNA methylation during fetal brain development were significantly underrepresented in promoter regulatory regions but significantly overrepresented in regions flanking CpG islands (shores and shelves) and gene bodies. Highly significant differences in DNA methylation were observed between males and females at a number of autosomal sites, with a small number of regions showing sex-specific DNA methylation trajectories across brain development. Weighted gene comethylation network analysis (WGCNA) revealed discrete modules of comethylated loci associated with fetal age that are significantly enriched for genes involved in neurodevelopmental processes. This is, to our knowledge, the most extensive study of DNA methylation across human fetal brain development to date, confirming the prenatal period as a time of considerable epigenomic plasticity. PMID:25650246

  12. Methylomic trajectories across human fetal brain development

    OpenAIRE

    Spiers, Helen; Hannon, Eilis; Schalkwyk, Leonard C; Smith, Rebecca; Wong, Chloe C. Y.; Michael C. O’Donovan; Bray, Nicholas J.; Mill, Jonathan

    2015-01-01

    Epigenetic processes play a key role in orchestrating transcriptional regulation during development. The importance of DNA methylation in fetal brain development is highlighted by the dynamic expression of de novo DNA methyltransferases during the perinatal period and neurodevelopmental deficits associated with mutations in the methyl-CpG binding protein 2 (MECP2) gene. However, our knowledge about the temporal changes to the epigenome during fetal brain development has, to date, been limited...

  13. Cell-free translation of murine coronavirus RNA.

    OpenAIRE

    Leibowitz, J L; Weiss, S.R.; Paavola, E; Bond, C W

    1982-01-01

    The coding assignments of the intracellular murine hepatitis virus-specific subgenomic RNA species and murine hepatitis virion RNA have been investigated by cell-free translation. The six murine hepatitis virus-specific subgenomic RNAs were partially purified by agarose gel electrophoresis and translated in an mRNA-dependent rabbit reticulocyte lysate, and the cell-free translation products were characterized by gel electrophoresis, immunoprecipitation, and tryptic peptide mapping. These stud...

  14. Fetal Alcohol Syndrome

    Science.gov (United States)

    ... Condiciones Chinese Conditions Fetal Alcohol Syndrome Read in Chinese What is Fetal Alcohol Syndrome (FAS)? Fetal Alcohol Syndrome (FAS) describes changes in a baby born to a mother whose pregnancy was complicated by alcohol consumption. A broader term ...

  15. Non-invasive prenatal diagnosis for fetal sex determination: benefits and disadvantages from the service users' perspective.

    Science.gov (United States)

    Lewis, Celine; Hill, Melissa; Skirton, Heather; Chitty, Lyn S

    2012-11-01

    Prenatal fetal sex determination is clinically indicated for women who are at risk of having a child with a serious genetic disorder affecting a particular sex. Ultrasound has been the traditional method used, but early fetal sex determination using non-invasive prenatal diagnosis (NIPD) can now be performed using cell-free fetal DNA in maternal plasma. The study aim was to assess the views and experiences of service users who had used NIPD for fetal sex determination. In this paper, we report on the perceived benefits and disadvantages. A qualitative approach using semi-structured interviews was used. A total of 44 participants (38 women and 6 partners of participating women) were recruited. Participants' views and experiences of NIPD were overwhelmingly positive. Concerning benefits over traditional methods, three themes emerged: (1) technical aspects of technology; (2) timing; and (3) enhanced decision-making. Practical advantages of NIPD included avoiding miscarriage, and there were a number of psychological advantages associated with timing such as perceived control, early re-engagement, normalization of pregnancy and peace of mind. Participants also valued NIPD as it enabled a stepwise approach to decision-making. A number of disadvantages were discussed including concerns about social sexing and increased bonding at a time in pregnancy when miscarriage risk is high. However, participants felt these were fairly minor in comparison with the advantages of NIPD. Until definitive genetic diagnosis using NIPD is available, NIPD for fetal sex determination is perceived as a good interim measure with a number of notable advantages over traditional methods. PMID:22453293

  16. A cell-free expression and purification process for rapid production of protein biologics.

    Science.gov (United States)

    Sullivan, Challise J; Pendleton, Erik D; Sasmor, Henri H; Hicks, William L; Farnum, John B; Muto, Machiko; Amendt, Eric M; Schoborg, Jennifer A; Martin, Rey W; Clark, Lauren G; Anderson, Mark J; Choudhury, Alaksh; Fior, Raffaella; Lo, Yu-Hwa; Griffey, Richard H; Chappell, Stephen A; Jewett, Michael C; Mauro, Vincent P; Dresios, John

    2016-02-01

    Cell-free protein synthesis has emerged as a powerful technology for rapid and efficient protein production. Cell-free methods are also amenable to automation and such systems have been extensively used for high-throughput protein production and screening; however, current fluidic systems are not adequate for manufacturing protein biopharmaceuticals. In this work, we report on the initial development of a fluidic process for rapid end-to-end production of recombinant protein biologics. This process incorporates a bioreactor module that can be used with eukaryotic or prokaryotic lysates that are programmed for combined transcription/translation of an engineered DNA template encoding for specific protein targets. Purification of the cell-free expressed product occurs through a series of protein separation modules that are configurable for process-specific isolation of different proteins. Using this approach, we demonstrate production of two bioactive human protein therapeutics, erythropoietin and granulocyte-macrophage colony-stimulating factor, in yeast and bacterial extracts, respectively, each within 24 hours. This process is flexible, scalable and amenable to automation for rapid production at the point-of-need of proteins with significant pharmaceutical, medical, or biotechnological value. PMID:26427345

  17. A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery.

    Science.gov (United States)

    Karim, Ashty S; Jewett, Michael C

    2016-07-01

    Speeding up design-build-test (DBT) cycles is a fundamental challenge facing biochemical engineering. To address this challenge, we report a new cell-free protein synthesis driven metabolic engineering (CFPS-ME) framework for rapid biosynthetic pathway prototyping. In our framework, cell-free cocktails for synthesizing target small molecules are assembled in a mix-and-match fashion from crude cell lysates either containing selectively enriched pathway enzymes from heterologous overexpression or directly producing pathway enzymes in lysates by CFPS. As a model, we apply our approach to n-butanol biosynthesis showing that Escherichia coli lysates support a highly active 17-step CoA-dependent n-butanol pathway in vitro. The elevated degree of flexibility in the cell-free environment allows us to manipulate physiochemical conditions, access enzymatic nodes, discover new enzymes, and prototype enzyme sets with linear DNA templates to study pathway performance. We anticipate that CFPS-ME will facilitate efforts to define, manipulate, and understand metabolic pathways for accelerated DBT cycles without the need to reengineer organisms. PMID:26996382

  18. Fetal syringomyelia.

    Science.gov (United States)

    Guo, Anne; Chitayat, David; Blaser, Susan; Keating, Sarah; Shannon, Patrick

    2014-01-01

    We explored the prevalence of syringomyelia in a series of 113 cases of fetal dysraphism and hindbrain crowding, of gestational age ranging from 17.5 to 34 weeks with the vast majority less than 26 weeks gestational age. We found syringomyelia in 13 cases of Chiari II malformations, 5 cases of Omphalocele/Exostrophy/Imperforate anus/Spinal abnormality (OEIS), 2 cases of Meckel Gruber syndrome and in a single pair of pyopagus conjoined twins. Secondary injury was not uncommon, with vernicomyelia in Chiari malformations, infarct like histology, or old hemorrhage in 8 cases of syringomyelia. Vernicomyelia did not occur in the absence of syrinx formation. The syringes extended from the sites of dysraphism, in ascending or descending patterns. The syringes were usually in a major proportion anatomically distinct from a dilated or denuded central canal and tended to be dorsal and paramedian or median. We suggest that fetal syringomyelia in Chiari II malformation and other dysraphic states is often established prior to midgestation, has contributions from the primary malformation as well as from secondary in utero injury and is anatomically and pathophysiologically distinct from post natal syringomyelia secondary to hindbrain crowding. PMID:25092126

  19. Maternal Plasma DNA and RNA Sequencing for Prenatal Testing.

    Science.gov (United States)

    Tamminga, Saskia; van Maarle, Merel; Henneman, Lidewij; Oudejans, Cees B M; Cornel, Martina C; Sistermans, Erik A

    2016-01-01

    Cell-free DNA (cfDNA) testing has recently become indispensable in diagnostic testing and screening. In the prenatal setting, this type of testing is often called noninvasive prenatal testing (NIPT). With a number of techniques, using either next-generation sequencing or single nucleotide polymorphism-based approaches, fetal cfDNA in maternal plasma can be analyzed to screen for rhesus D genotype, common chromosomal aneuploidies, and increasingly for testing other conditions, including monogenic disorders. With regard to screening for common aneuploidies, challenges arise when implementing NIPT in current prenatal settings. Depending on the method used (targeted or nontargeted), chromosomal anomalies other than trisomy 21, 18, or 13 can be detected, either of fetal or maternal origin, also referred to as unsolicited or incidental findings. For various biological reasons, there is a small chance of having either a false-positive or false-negative NIPT result, or no result, also referred to as a "no-call." Both pre- and posttest counseling for NIPT should include discussing potential discrepancies. Since NIPT remains a screening test, a positive NIPT result should be confirmed by invasive diagnostic testing (either by chorionic villus biopsy or by amniocentesis). As the scope of NIPT is widening, professional guidelines need to discuss the ethics of what to offer and how to offer. In this review, we discuss the current biochemical, clinical, and ethical challenges of cfDNA testing in the prenatal setting and its future perspectives including novel applications that target RNA instead of DNA. PMID:27117661

  20. Cell-free protein production for NMR studies.

    Science.gov (United States)

    Takeda, Mitsuhiro; Kainosho, Masatsune

    2012-01-01

    The cell-free expression system using an Escherichia coli extract is a practical method for producing isotope-labeled proteins. The advantage of the cell-free system over cellular expression is that any isotope-labeled amino acid can be incorporated into the target protein with minimal scrambling, thus providing opportunities for advanced isotope labeling of proteins. We have modified the standard protocol for E. coli cell-free expression to cope with two problems specific to NMR sample preparation. First, endogenous amino acids present in the E. coli S30 extract lead to dilution of the added isotope. To minimize the content of the remaining amino acids, a gel filtration step is included in the preparation of the E. coli extract. Second, proteins produced by the cell-free system are not necessarily homogeneous due to incomplete processing of the N-terminal formyl-methionine residue, which complicates NMR spectra. Therefore, the protein of interest is engineered to contain a cleavable N-terminal histidine-tag, which generates a homogeneous protein after the digestion of the tag. Here, we describe the protocol for modified E. coli cell-free expression. PMID:22167669

  1. Tissue-specific transcription enhancement of the fibroin gene characterized by cell-free systems.

    OpenAIRE

    Suzuki, Y.; Tsuda, M.; Takiya, S; Hirose, S; Suzuki, E; Kameda, M; Ninaki, O

    1986-01-01

    Six cell-free extracts have been used to characterize the nature of DNA signals and trans-acting factors responsible for the transcription enhancement of the Bombyx mori fibroin gene. The upstream element of the fibroin gene involved in the enhancement can be divided into two regions. The proximal region, -72 to -32, is recognized as a common enhancing signal by all B. mori extracts from the posterior silk gland, the middle silk gland, the ovarian tissue, and an embryonic cell line. It is wea...

  2. Medio ambiente fetal Fetal environment

    Directory of Open Access Journals (Sweden)

    César Bernardo Ospina Arcila

    1996-04-01

    Full Text Available Con base en el artículo clásico "Monte Everest in utero" se hace un análisis de la situación que afronta el feto con respecto a la disponibilidad de oxígeno; para una mejor comprensión del sufrimiento fetal se revisan los siguientes conceptos: presión barométrica, presión parcial del oxígeno atmosférico, presión parcial del oxígeno inspirado, presión barométrica intranasal, ecuación del gas alveolar y difusión de gases a través de la membrana alvéolo capilar. Based on the classical paper by Eastman "Mount Everest in utero" an analysis is made of the situation faced by the fetus with respect to the availability of oxygen; for a better under. standing of fetal distress the following concepts are reviewed: barometric pressure, partial pressure of atmosferic oxygen, partial pressure of inspired oxygen, barometric intranasal pressure, alveolar gas equation and gas diffusion through alveolo-capilar membrane.

  3. Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing.

    Science.gov (United States)

    Ståhlberg, Anders; Krzyzanowski, Paul M; Jackson, Jennifer B; Egyud, Matthew; Stein, Lincoln; Godfrey, Tony E

    2016-06-20

    Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing library construction with flexible library content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants. PMID:27060140

  4. Radio-modification by caffeine alone and in combination with phosphorothioates: in vivo and cell-free studies

    International Nuclear Information System (INIS)

    Caffeine is generally considered to result in radiosensitization by affecting the cell cycle. Data from in vivo studies, however, do not suggest sensitization; caffeine administration did not adversely affect survival of mice irradiated at doses causing hematopoietic injury, or gastrointestinal injury, or when administered in combination with phosphorothioates. For example, caffeine administration (20 mg/kg IP) in combination with the radioprotector WR-151327, S-2-(3-methyl-amino-propyl-amino)propyl-phosphoro-thioic acid. (200 mg/kg IP) resulted in a dose modification factor of 1.54 in comparison to 1.51 for WR-151327 treatment alone. In a cell-free system, the active metabolites of phosphorothiotates, i.e. free thiols and disulfides, appear to mimic polyamines and modulate enzymes involves in DNA structure and synthesis. The free thiol of WR-151327 (WR-151326) actively enhanced topoisomerase I-mediated unwinding of supercoiled plB130 DNA and super-coiling of DNA mediated by DNA gyrase (topoisomerase II). Caffeine, in general, had opposite effects on potoisomerase activities compared to WR-151326. When caffeine was added to the cell-free system together with WR-151326, the stimulatory effects of WR-151326 were suppressed. Further studies are needed in cell-free systems, cells, and animals to elucidate the potential utility of caffeine administration in combination with radiation and other therapeutic agents. (authors)

  5. Application evaluation of noninvasive DNA detection in the prenatal screening of fetal Down syndrome%无创DNA检测在胎儿唐氏综合征产前筛查中的应用评价

    Institute of Scientific and Technical Information of China (English)

    王慧; 熊敏; 韩宝良; 江德洋

    2016-01-01

    目的:评价无创 DNA 检测在诊断唐氏综合征中的应用价值。方法:807例单胎孕妇分为高龄组、高危组、中危组,进行无创DNA检测,得出胎儿发生唐氏综合征的风险率。结果:高龄组121例孕妇中21三体阳性3例,高危组388例中4例,中危组298例中0例。7例阳性者经羊水穿刺及胎儿染色体核型分析证实为21三体阳性。625例阴性者出生后无唐氏综合征。结论:产前孕妇外周血中胎儿游离DNA检测对于唐氏综合征的产前诊断具有创伤小、安全、检出率及准确率高等优点。%Objective:To evaluate the application value of noninvasive DNA detection in the diagnosis of Down syndrome. Methods:807 cases of singleton pregnant women were divided into the elderly group,the high risk group,the middle risk group. They were given noninvasive DNA detection.The risk ratio of fetal Down syndrome was obtained.Results:3 cases were 21 three body positive in 121 pregnant women of the elderly group.4 cases were positive in 388 cases of the high risk group.0 case was positive in 298 cases of the middle risk group.7 positive cases were three body positive that were confirmed by amniotic fluid puncture and fetal karyotype analysis.625 negative cases had no Down syndrome after birth.Conclusion:Prenatal fetal free DNA detection in peripheral blood of pregnant women has the advantages of small trauma,safety,high detection rate and accuracy rate in the prenatal diagnosis of Down syndrome.

  6. Pathophysiological consequences of hemolysis. Role of cell-free hemoglobin

    Directory of Open Access Journals (Sweden)

    Tomasz Misztal

    2011-09-01

    Full Text Available Abundant hemolysis is associated with a number of inherent and acquired diseases including sickle-cell disease (SCD, polycythemia, paroxysmal nocturnal hemoglobinuria (PNH and drug-induced hemolytic anemia. Despite different etiopathology of hemolytic diseases, many concomitant symptoms are comparable and include e.g. hypertension, hemoglobinuria and hypercoagulation state. Studies in the last years have shown a growing list of mechanisms lying at the basis of those symptoms, in particular irreversible reaction between cell-free hemoglobin (Hb and nitric oxide (NO – endogenous vasorelaxant and anti-thrombotic agent. Saturation of protective physiological cell-free Hb-scavenging mechanisms results in accumulation of Hb in plasma and hemoglobinemia. Extensive hemoglobinemia subsequently leads to hemoglobinuria, which may cause kidney damage and development of Fanconi syndrome. A severe problem in patients with SCD and PNH is pulmonary and systemic hypertension. It may lead to circulation failure, including stroke, and it is related to abolition of NO bioavailability for vascular smooth muscle cells. Thrombotic events are the major cause of death in SCD and PNH. It ensues from lack of platelet inhibition evoked by Hb-mediated NO scavenging. A serious complication that affects patients with excessive hemolysis is erectile dysfunction. Also direct cytotoxic, prooxidant and proinflammatory effects of cell-free hemoglobin and heme compose the clinical picture of hemolytic diseases. The pathophysiological role of plasma Hb, mechanisms of its elimination, and direct and indirect (via NO scavenging deleterious effects of cell-free Hb are presented in detail in this review. Understanding the critical role of hemolysis and cell-free Hb is important in the perspective of treating patients with hemolytic diseases and to design new effective therapies in future.

  7. Studies on the isolation, structural analysis and tissue localization of fetal antigen 1 and its relation to a human adrenal-specific cDNA, pG2

    DEFF Research Database (Denmark)

    Jensen, Charlotte Harken; Teisner, Børge; Højrup, Peter;

    1993-01-01

    sequence was back-translated into the appropriate degenerate sequence of nucleic acids, fetal antigen 1 could be partially aligned to a 'human adrenal-specific mRNA, pG2'. The indirect immunoperoxidase technique demonstrated fetal antigen 1 in fetal hepatocytes, glandular cells of fetal pancreas and in...

  8. Cell-free system for synthesizing membrane proteins cell free method for synthesizing membrane proteins

    Science.gov (United States)

    Laible, Philip D; Hanson, Deborah K

    2013-06-04

    The invention provides an in vitro method for producing proteins, membrane proteins, membrane-associated proteins, and soluble proteins that interact with membrane-associated proteins for assembly into an oligomeric complex or that require association with a membrane for proper folding. The method comprises, supplying intracytoplasmic membranes from organisms; modifying protein composition of intracytoplasmic membranes from organism by modifying DNA to delete genes encoding functions of the organism not associated with the formation of the intracytoplasmic membranes; generating appropriate DNA or RNA templates that encode the target protein; and mixing the intracytoplasmic membranes with the template and a transcription/translation-competent cellular extract to cause simultaneous production of the membrane proteins and encapsulation of the membrane proteins within the intracytoplasmic membranes.

  9. Reduced DNA methylation at the PEG3 DMR and KvDMR1 loci in children exposed to alcohol in utero: a South African Fetal Alcohol Syndrome cohort study.

    Science.gov (United States)

    Masemola, Matshane L; van der Merwe, Lize; Lombard, Zané; Viljoen, Denis; Ramsay, Michèle

    2015-01-01

    Fetal alcohol syndrome (FAS) is a devastating developmental disorder resulting from alcohol exposure during fetal development. It is a considerable public health problem worldwide and is characterized by central nervous system abnormalities, dysmorphic facial features, and growth retardation. Imprinted genes are known to play an important role in growth and development and therefore four imprinting control regions (ICRs), H19 ICR, IG-DMR, KvDMR1 and PEG3 DMR were examined. It is proposed that DNA methylation changes may contribute to developmental abnormalities seen in FAS and which persist into adulthood. The participants included FAS children and controls from the Western and Northern Cape Provinces. DNA samples extracted from blood and buccal cells were bisulfite modified, the ICRs were amplified by PCR and pyrosequencing was used to derive a quantitative estimate of methylation at selected CpG dinucleotides: H19 ICR (six CpG sites; 50 controls and 73 cases); KvDMR1 (7, 55, and 86); IG-DMR (10, 56, and 84); and PEG3 DMR (7, 50, and 79). The most profound effects of alcohol exposure are on neuronal development. In this study we report on epigenetic effects observed in blood which may not directly reflect tissue-specific alterations in the developing brain. After adjusting for age and sex (known confounders for DNA methylation), there was a significant difference at KvDMR1 and PEG3 DMR, but not the H19 ICR, with only a small effect (0.84% lower in cases; p = 0.035) at IG-DMR. The two maternally imprinted loci, KvDMR1 and PEG3 DMR, showed lower average locus-wide methylation in the FAS cases (1.49%; p alcohol by altering the methylation profiles of imprinted loci in a locus-specific manner. PMID:25806045

  10. Investigating and correcting plasma DNA sequencing coverage bias to enhance aneuploidy discovery.

    Directory of Open Access Journals (Sweden)

    Dineika Chandrananda

    Full Text Available Pregnant women carry a mixture of cell-free DNA fragments from self and fetus (non-self in their circulation. In recent years multiple independent studies have demonstrated the ability to detect fetal trisomies such as trisomy 21, the cause of Down syndrome, by Next-Generation Sequencing of maternal plasma. The current clinical tests based on this approach show very high sensitivity and specificity, although as yet they have not become the standard diagnostic test. Here we describe improvements to the analysis of the sequencing data by reducing GC bias and better handling of the genomic repeats. We show substantial improvements in the sensitivity of the standard trisomy 21 statistical tests, which we measure by artificially reducing read coverage. We also explore the bias stemming from the natural cleavage of plasma DNA by examining DNA motifs and position specific base distributions. We propose a model to correct this fragmentation bias and observe that incorporating this bias does not lead to any further improvements in the detection of fetal trisomy. The improved bias corrections that we demonstrate in this work can be readily adopted into existing fetal trisomy detection protocols and should also lead to improvements in sub-chromosomal copy number variation detection.

  11. Cell-free protein synthesis and purification of human dopamine D2 receptor long isoform.

    Science.gov (United States)

    Basu, Dipannita; Castellano, Jessica M; Thomas, Nancy; Mishra, Ram K

    2013-01-01

    The human dopamine D2 receptor long isoform (D2L) has significant implications in neurological and neuropsychiatric disorders such as Parkinson's disease and schizophrenia. Detailed structural knowledge of this receptor is limited owing to its highly hydrophobic nature, which leads to protein aggregation and host toxicity when expressed in cellular systems. The newly emerging field of cell-free protein expression presents numerous advantages to overcome these challenges. This system utilizes protein synthesis machinery and exogenous DNA to synthesize functional proteins outside of intact cells. This study utilizes two different cell-free systems for the synthesis of human dopamine D2L receptor. These include the Escherichia coli lysate-based system and the wheat-germ lysate-based system. The bacterial cell-free method used pET 100/D-TOPO vector to synthesize hexa-histidine-tagged D2L receptor using a dialysis bag system; the resulting protein was purified using nickel-nitrilotriacetic acid affinity resin. The wheat germ system used pEU-glutathione-S-transferase (GST) vector to synthesize GST-tagged D2L receptor using a bilayer translation method; the resulting protein was purified using a GST affinity resin. The presence and binding capacity of the synthesized D2L receptor was confirmed by immunoblotting and radioligand competition assays, respectively. Additionally, in-gel protein sequencing via Nano LC-MS/MS was used to confirm protein synthesis via the wheat germ system. The results showed both systems to synthesize microgram quantities of the receptor. Improved expression of this highly challenging protein can improve research and understanding of the human dopamine D2L receptor. PMID:23424095

  12. Fetal Alcohol Spectrum Disorders

    Science.gov (United States)

    ... Daily life skills, such as feeding and bathing Fetal alcohol syndrome is the most serious type of FASD. People with fetal alcohol syndrome have facial abnormalities, including wide-set and narrow ...

  13. Fetal Alcohol Exposure

    Science.gov (United States)

    ... a woman drinks while pregnant. Alcohol can disrupt fetal development at any stage during a pregnancy—including at ... Clinical Diagnoses IOM Diagnoses Fetal Alcohol Syndrome ... pregnancy can disrupt normal development of the face and the brain. In fact, ...

  14. Cell-Free Metabolic Engineering: Biomanufacturing beyond the cell

    OpenAIRE

    Dudley, Quentin M.; Karim, Ashty S.; Jewett, Michael C.

    2014-01-01

    Industrial biotechnology and microbial metabolic engineering are poised to help meet the growing demand for sustainable, low-cost commodity chemicals and natural products, yet the fraction of biochemicals amenable to commercial production remains limited. Common problems afflicting the current state-of-the-art include low volumetric productivities, build-up of toxic intermediates or products, and byproduct losses via competing pathways. To overcome these limitations, cell-free metabolic engin...

  15. Cell-free translation of bovine viral diarrhea virus RNA.

    OpenAIRE

    Purchio, A F; Larson, R.; Torborg, L L; Collett, M S

    1984-01-01

    Bovine viral diarrhea virus RNA was translated in a reticulocyte cell-free protein synthesizing system. The purified, 8.2-kilobase, virus-specific RNA species was unable to serve an an efficient message unless it was denatured immediately before translation. In this case, several polypeptides, ranging in molecular weight from 50,000 to 150,000 and most of which were immunoprecipitated by bovine viral diarrhea virus-specific antiserum, were synthesized in vitro. When polyribosomes were used to...

  16. Overview of Production of Protein Using Cell-free Systems

    OpenAIRE

    Gao, Fei Philip

    2014-01-01

    One of the most important steps in protein research is production of the target protein. Cell based systems are mature tools that have long been used to express recombinant proteins by manipulation of the expression organisms. However, it is often challenging to find suitable cell systems that allow for rapid screening of conditions and constructs to produce properly folded, functional proteins in a cost effective manner. As a result, cell-free protein production emerged as an attractive alte...

  17. Radionuclide fetal imaging

    International Nuclear Information System (INIS)

    Hepato-biliary scanning with sup (99m) Tc-P.G. demonstrated in a jaundiced patient at 31 weeks pregnancy the filling of the fetal bladder 6 h after injection. This finding was confirmed by ultrasound. Fetal urinary bladder activity was seen until 33 h after injection. Fetal hepatic radio-activity could not be displayed although the localisation of the fetal liver was marked by ultrasound. (orig.)

  18. Cell-Free Synthesis Meets Antibody Production: A Review

    Directory of Open Access Journals (Sweden)

    Marlitt Stech

    2015-01-01

    Full Text Available Engineered antibodies are key players in therapy, diagnostics and research. In addition to full size immunoglobulin gamma (IgG molecules, smaller formats of recombinant antibodies, such as single-chain variable fragments (scFv and antigen binding fragments (Fab, have emerged as promising alternatives since they possess different advantageous properties. Cell-based production technologies of antibodies and antibody fragments are well-established, allowing researchers to design and manufacture highly specific molecular recognition tools. However, as these technologies are accompanied by the drawbacks of being rather time-consuming and cost-intensive, efficient and powerful cell-free protein synthesis systems have been developed over the last decade as alternatives. So far, prokaryotic cell-free systems have been the focus of interest. Recently, eukaryotic in vitro translation systems have enriched the antibody production pipeline, as these systems are able to mimic the natural pathway of antibody synthesis in eukaryotic cells. This review aims to overview and summarize the advances made in the production of antibodies and antibody fragments in cell-free systems.

  19. Fetal behavioral teratology

    NARCIS (Netherlands)

    Visser, Gerard H. A.; Mulder, Eduard J. H.; Ververs, F. F. Tessa

    2010-01-01

    Ultrasound studies of fetal motor behavior provide direct - in vivo - insight in the functioning of the motor component of the fetal central nervous system. In this article, studies are reviewed showing changes in the first timetable of appearance of fetal movements, changes in quality and/or quanti

  20. Absence of regulation of tumor cholesterogenesis in cell-free synthesizing systems

    International Nuclear Information System (INIS)

    In tumors, cholesterol synthesis de novo is deregulated relative to normal tissues. But no previous study has demonstrated the decontrol of tumor cholesterogenesis with cell-free cytosolic systems. They have utilized a lipid synthesizing, post-mitochondrial supernatant system (PMS), with 14C-citrate as substrate, to characterize the cholesterogenic pathway in Morris Hepatoma 3924A and normal rat liver. The rate of cholesterogenesis in the hepatoma PMS was 6-fold higher than that in the liver system on a per cell basis. The ratio of sterol-to-fatty acid synthesis was also significantly greater in the tumor versus the liver PMS. The authors determined the steady-state carbon flux through the early intermediates of the lipogenic pathways. Whereas the liver system displayed a metabolic crossover point at the HMG-CoA reductase reaction, the hepatoma system showed no evidence of control at this rate-limiting site of sterol synthesis. Furthermore, acetyl-CoA formation from added citrate (via ATP-citrate lyase) exhibited rates of 42% and 88% in excess of that required for lipidogenesis by liver and tumor PMS systems, respectively. Clearly, a cell-free PMS system from tumor tissue displays the property of deregulated lipidogenesis, especially cholesterol biosynthesis. The authors suggest that deregulated and continuously operating cholesterogenesis would provide for an increased level of a mevalonate-derived sterol pathway intermediate proposed as a trigger for DNA synthesis and cell proliferation in tumors

  1. Efficient production of isotopically labeled proteins by cell-free synthesis: A practical protocol

    International Nuclear Information System (INIS)

    We provide detailed descriptions of our refined protocols for the cell-free production of labeled protein samples for NMR spectroscopy. These methods are efficient and overcome two critical problems associated with the use of conventional Escherichia coli extract systems. Endogenous amino acids normally present in E. coli S30 extracts dilute the added labeled amino acids and degrade the quality of NMR spectra of the target protein. This problem was solved by altering the protocol used in preparing the S30 extract so as to minimize the content of endogenous amino acids. The second problem encountered in conventional E. coli cell-free protein production is non-uniformity in the N-terminus of the target protein, which can complicate the NMR spectra. This problem was solved by adding a DNA sequence to the construct that codes for a cleavable N-terminal peptide tag. Addition of the tag serves to increase the yield of the protein as well as to ensure a homogeneous protein product following tag cleavage. We illustrate the method by describing its stepwise application to the production of calmodulin samples with different stable isotope labeling patterns for NMR analysis

  2. Plant RNA processing: soybean pre-mRNA in a pea cell-free extract

    International Nuclear Information System (INIS)

    Using a pea cell-free extract they have demonstrated the splicing of an SP6 fusion transcript containing an intron derived from the soybean seed storage protein β-subunit gene. Intron 115 from the conglycinin gene was cloned into a SP6 vector and transcribed using standard recombinant DNA techniques. Incubation of radioactively labeled fusion transcripts in the cell-free system produced a number of products which were identified by primer extension and S1 nuclease analysis. All the products are linear RNA molecules. Lariat intermediates, similar to those found in the yeast and HeLa cell RNA processing systems, have not been detected. The linear RNA products detected in their plant in vitro processing system have various portions of the intron removed which suggests that alternative splice sites are used in processing of this plant intron due to activation of cryptic splice sites or creation of splice sites in the fusion construction. The kinetics of the reactions and parameters of the extract are similar to those determined for the HeLa cell system. Sucrose gradient analysis has demonstrated that the plant RNA products sedimented in a 30S particle, similar in size to that found for the spliceosome of the HeLa cell system

  3. Hemodynamics in fetal arrhythmia.

    Science.gov (United States)

    Sonesson, Sven-Erik; Acharya, Ganesh

    2016-06-01

    Fetal arrhythmias are among the few conditions that can be managed in utero. However, accurate diagnosis is essential for appropriate management. Ultrasound-based imaging methods can be used to study fetal heart structure and function noninvasively and help to understand fetal cardiovascular pathophysiology, and they remain the mainstay of evaluating fetuses with arrhythmias in clinical settings. Hemodynamic evaluation using Doppler echocardiography allows the elucidation of the electrophysiological mechanism and helps to make an accurate diagnosis. It can also be used as a tool to understand fetal cardiac pathophysiology, for assessing fetal condition and monitoring the effect of antiarrhythmic treatment. This narrative review describes Doppler techniques that are useful for evaluating fetal cardiac rhythms to refine diagnosis and provides an overview of hemodynamic changes observed in different types of fetal arrhythmia. PMID:26660845

  4. Differing levels of excision repair in human fetal dermis and brain cells

    Energy Technology Data Exchange (ETDEWEB)

    Gibson, R.E. (Ohio State Univ., Columbus (USA). Dept. of Radiology); D' Ambrosio, S.M. (Ohio State Univ., Columbus (USA). Dept. of Radiology; Ohio State Univ., Columbus (USA). Dept. of Pharmacology)

    1982-01-01

    The levels of DNA excision repair, as measured by unscheduled DNA synthesis (UDS) and the UV-endonuclease sensitive site assay, were compared in cells derived from human fetal brain and dermal tissues. The level of UDS induced following ultraviolet (UV) irradiation was found to be lower (approx. 60%) in the fetal brain cells than in fetal dermal cells. It was determined, using the UV-endonuclease sensitive site assay to confirm the UDS observation, that 50% of the dimers induced by UV in fetal dermal cells were repaired in 8 h. while only 15% were removed in the fetal brain cells during the same period of time. Even after 24 h. only 44% of the dimers induced by UV in the fetal brain cells were repaired, while 65% were removed in the dermal cells. These data suggest that cultured human fetal brain cells exhibit lower levels of excision repair compared to cultured human fetal dermal cells.

  5. Cell-free nucleic acids as a non-invasive route for investigating atherosclerosis.

    Science.gov (United States)

    Cerne, Darko; Bajalo, Jana Lukac

    2014-01-01

    Metabolic syndrome is directly linked with atherosclerotic burden and cell-free nucleic acids (cf-NA) analysis has recently emerged as a novel research tool in atherosclerosis practice and research. cf-NA are nucleic acids (DNA, mRNA, miRNA, mitochondrial DNA) found in plasma and cell-free fractions of various other biological fluids. They have all the characteristics of the nucleic acids in the cells of their origin, thus constituting an emerging field for non-invasive assessment. Initially, quantitative and qualitative analysis of cf-NA has been accepted as clinically useful in non-invasive prenatal diagnosis, and in the diagnosis and monitoring of numerous cancers. As to atherosclerosis, cf-NA analysis poses an important challenge in diagnosis and prognostic evaluation of acute coronary syndrome, in prediction of cardiovascular disease, in non-invasive early detection of atherosclerosis and understanding its pathological mechanism in vivo, in assessing various issues of treatment for atherosclerosis in vivo, and in the unique simultaneous measurement of mRNA levels and protein concentrations in a single sample of plasma. Examples of its use are presented in this review. Besides the advances in technologies, the precise evaluation and optimization of pre-analytical and analytical aspects of cf-NA analysis have impacted importantly on the reliability of test results. We have, therefore, reviewed the most important analytical considerations. Further clinical studies and analytical improvements will answer the question as to whether cf-NA, as novel biomarkers, can be reliably applied clinically in non-invasive, early diagnosis and monitoring of the vulnerable atherosclerotic plaques of patients who could suffer from acute coronary syndrome. PMID:24320033

  6. Cell-free metabolic engineering: Biomanufacturing beyond the cell

    Energy Technology Data Exchange (ETDEWEB)

    Dudley, QM; Karim, AS; Jewett, MC

    2014-10-15

    Industrial biotechnology and microbial metabolic engineering are poised to help meet the growing demand for sustainable, low-cost commodity chemicals and natural products, yet the fraction of biochemicals amenable to commercial production remains limited. Common problems afflicting the current state-of-the-art include low volumetric productivities, build-up of toxic intermediates or products, and byproduct losses via competing pathways. To overcome these limitations, cell-free metabolic engineering (CFME) is expanding the scope of the traditional bioengineering model by using in vitro ensembles of catalytic proteins prepared from purified enzymes or crude lysates of cells for the production of target products. In recent years, the unprecedented level of control and freedom of design, relative to in vivo systems, has inspired the development of engineering foundations for cell-free systems. These efforts have led to activation of long enzymatic pathways (>8 enzymes), near theoretical conversion yields, productivities greater than 100 mg L-1 h(-1), reaction scales of >100 L, and new directions in protein purification, spatial organization, and enzyme stability. In the coming years, CFME will offer exciting opportunities to: (i) debug and optimize biosynthetic pathways; (ii) carry out design-build-test iterations without re-engineering organisms; and (iii) perform molecular transformations when bioconversion yields, productivities, or cellular toxicity limit commercial feasibility.

  7. Fetal Health and Development

    Science.gov (United States)

    ... specific prenatal tests to monitor both the mother's health and fetal health during each trimester. With modern technology, health professionals can Detect birth defects Identify problems that ...

  8. Maternal kandan noninvazif yöntemlerle fetal Rh tayini

    OpenAIRE

    Gönenç, Gökçenur

    2013-01-01

    Amaç: Bu çalışmanın amacı Rh uygunsuzluğu olan gebelerde fetal Rh durumunun invaziv olmayan bir teknikle, maternal kandan serbest fetal DNA izolasyonu ile tespit edilmesidir. Özellikle daha önceki gebeliklerinde fetal anemi gelişen gebelerde fetusun RhD pozitif olduğunun bilinmesi fetusun yakın takibine olanak verecektir. Yöntem: Rh uygunsuzluğu olan 50 gebeden serbest fetal DNA izolasyonu yapıldı. İzole edilen DNA, TaqMan probu ve RhD geninin exon 7 bölgesine ait primerlerle Real Time P...

  9. Fetal Kidney Anomalies: Next Generation Sequencing

    DEFF Research Database (Denmark)

    Rasmussen, Maria; Sunde, Lone; Nielsen, Marlene Louise;

    undergone postmortem examination. The approximately 110 genes included in the targeted panel were chosen on the basis of their potential involvement in embryonic kidney development, cystic kidney disease, or the renin-angiotensin system. DNA was extracted from fetal tissue samples or cultured chorion villus...... the nephronophthisis associated gene, TMEM67 and six fetuses had mutations in kidney developmental genes. For these fetuses kidney histology is presented. Conclusion and Perspectives In eight (14%) fetuses we identified a likely genetic cause of the kidney anomalies. Ten fetuses from eight families......, in which no mutations were identified, have been selected for exome sequencing in order to uncover novel genes associated to fetal kidney anomalies....

  10. Enterococcus faecium LKE12 Cell-Free Extract Accelerates Host Plant Growth via Gibberellin and Indole-3-Acetic Acid Secretion.

    Science.gov (United States)

    Lee, Ko-Eun; Radhakrishnan, Ramalingam; Kang, Sang-Mo; You, Young-Hyun; Joo, Gil-Jae; Lee, In-Jung; Ko, Jae-Hwan; Kim, Jin-Ho

    2015-09-01

    The use of microbial extracts containing plant hormones is a promising technique to improve crop growth. Little is known about the effect of bacterial cell-free extracts on plant growth promotion. This study, based on phytohormonal analyses, aimed at exploring the potential mechanisms by which Enterococcus faecium LKE12 enhances plant growth in oriental melon. A bacterial strain, LKE12, was isolated from soil, and further identified as E. faecium by 16S rDNA sequencing and phylogenetic analysis. The plant growth-promoting ability of an LKE12 bacterial culture was tested in a gibberellin (GA)-deficient rice dwarf mutant (waito-C) and a normal GA biosynthesis rice cultivar (Hwayongbyeo). E. faecium LKE12 significantly improved the length and biomass of rice shoots in both normal and dwarf cultivars through the secretion of an array of gibberellins (GA1, GA3, GA7, GA8, GA9, GA12, GA19, GA20, GA24, and GA53), as well as indole-3-acetic acid (IAA). To the best of our knowledge, this is the first study indicating that E. faecium can produce GAs. Increases in shoot and root lengths, plant fresh weight, and chlorophyll content promoted by E. faecium LKE12 and its cell-free extract inoculated in oriental melon plants revealed a favorable interaction of E. faecium LKE12 with plants. Higher plant growth rates and nutrient contents of magnesium, calcium, sodium, iron, manganese, silicon, zinc, and nitrogen were found in cell-free extract-treated plants than in control plants. The results of the current study suggest that E. faecium LKE12 promotes plant growth by producing GAs and IAA; interestingly, the exogenous application of its cell-free culture extract can be a potential strategy to accelerate plant growth. PMID:25907061

  11. Challenge of Fetal Mortality

    Science.gov (United States)

    ... to yield the number of previous pregnancies. Data source and methods This report contains data from the Fetal Death Data File and Linked Birth/Infant Death Data Set, NVSS. The vital statistics Fetal Death Data File contains information from all ...

  12. Fetal Neurobehavioral Development.

    Science.gov (United States)

    DiPietro, Janet A.; And Others

    1996-01-01

    Investigated the ontogeny of fetal autonomic, motoric, state, and interactive functioning in 31 healthy fetuses from 20 weeks through term. Found that male fetuses were more active than female fetuses, and that greater maternal stress appraisal was associated with reduced fetal heart rate variability. Found that an apparent period of…

  13. Noninvasive prenatal diagnosis of β-thalassaemia using size -fractionated cell -free fetal DNA in matemal plasma.%利用孕妇外周血浆中小片段游离胎儿DNA进行β-地中海贫血无创性产前诊断

    Institute of Scientific and Technical Information of China (English)

    龙兴江; 龙桂芳; 林伟雄

    2008-01-01

    目的 利用cffDNA对胎儿进行广西、广东地区常见的17种β-地中海贫血基因型的检测,与传统创伤性产前诊断相比较,探讨该方法的准确性和可行性.方法 针对广西、广东地区常见的β-地中海贫血突变的基因型设计三对不同引物,并用生物素标记.对cffDNA进行二次PCR反应后,使用反向斑点杂交(revert dot-blot hybridization,RDB)检测胎儿的β-珠蛋白基因型,与创伤性产前诊断结果比较,观察准确率.结果 37例cffDNA标本检测中,检出重型β-地中海贫血19例,轻型β-地中海贫血11例,正常7例,与创伤性产前诊断结果相比较出现3例误诊,准确率为91.9%(34/37).结论 利用effDNA进行β-地中海贫血的检测,可能出现母源性DNA背景的污染,当胎儿基因型与母亲基因型相同时,必须提高警惕,进一步分析或者复查.因为该技术取样容易,对孕妇胎儿无风险,不受孕期时间影响,易为与孕妇接受,因此进一步改进该技术后有望可用于β-地中海贫血的诊断.

  14. Perspective study of non-invasive prenatal testing using cell-free fetal DNA in high-risk population%高危孕妇外周血胎儿游离核酸无创性产前检测的前瞻性研究

    Institute of Scientific and Technical Information of China (English)

    马京梅; 潘虹; 付杰; 于丽; 杨慧霞

    2015-01-01

    Objective To evaluate the application of non-invasive prenatal testing (NIPT) in high-risk pregnancies before invasive prenatal diagnosis.Methods This prospective study involved all pregnancies participating in the clinical trial of NIPT validation.The indication,age and gestational week were recorded.And the results of karyotype and NIPT risk were compared.Results A total of 2 439 high-risk pregnancies were enrolled from 18 prenatal diagnosis clinics in China.After excluding 48 cases (36 missing clinical information,9 karyotype failures and 3 NIPT failures),2 391 cases were analyzed.Their gestational ages were 10 to 31 weeks.And 56.3% (1 345/2 691) were at a high risk for mid-trimester screening,following by advanced maternal age (26.0%,621/2 391).Forty-six aneuploidies (trisomy 21,n =38 ; trisomy 18,n =7 ; trisomy 13,n =1) were diagnosed by karyotyping.And it was consistent with the NIPT results of high-risk distribution.Conclusion NIPT may be used to reduce unnecessary invasive prenatal diagnosis by improving the efficacy of current mid-trimester screening strategy,particularly for trisomy 21.%目的 评价无创性产前检测技术(NIPT)在准备进行侵入性产前诊断的高危孕妇中的应用.方法 2014年2至5月,前瞻性入组参与NIPT试剂盒临床试验的北京大学第一医院、浙江大学医学院附属妇产科医院及第四军医大学西京医院等,共18家产前诊断中心的高危孕妇,记录临床指征、年龄、孕周等数据,进行侵入性产前诊断染色体核型分析结果和NIPT结果对比.结果 18家产前诊断中心共2 439例高危孕妇入组,剔除36例临床纪录不全、9例染色体核型分析失败、3例NIPT检测失败病例,对2 391例病例进行分析.孕周范围11 ~31周,产前诊断指征以血清学筛查高风险为主,占56.3%(1 345/2 691),高龄孕妇26.0%(621/2 391).染色体核型分析结果共发现46例常见非整倍体(Trisomy,T)(38例T21,7例T18,1例T13),同期NIPT结果提示为一一对应T21、18、13高风险,同时未见“NIPT高风险而核型分析为正常”病例.结论 对于现有以中孕期血清学筛查高危为主要指征的产前诊断高危孕妇,通过NIPT可以大幅提高T21、T18、T13的筛查效率,从而减少不必要的侵入性产前诊断.

  15. Enhanced cell-free protein expression by fusion with immunoglobulin Cκ domain

    OpenAIRE

    Palmer, Elizabeth; Liu, Hong; Khan, Farid; Taussig, Michael J; He, Mingyue

    2006-01-01

    While cell-free systems are increasingly used for protein expression in structural and functional studies, several proteins are difficult to express or expressed only at low levels in cell-free lysates. Here, we report that fusion of the human immunoglobulin κ light chain constant domain (Cκ) at the C terminus of four representative proteins dramatically improved their production in the Escherichia coli S30 system, suggesting that enhancement of cell-free protein expression by Cκ fusion will ...

  16. Chromophore maturation and fluorescence fluctuation spectroscopy of fluorescent proteins in a cell-free expression system

    OpenAIRE

    Macdonald, Patrick J.; Chen, Yan; Mueller, Joachim D.

    2011-01-01

    Cell-free synthesis, a method for the rapid expression of proteins, is increasingly used to study interactions of complex biological systems. GFP and its variants have become indispensable for fluorescence studies in live cells and are equally attractive as reporters for cell-free systems. This work investigates the use of fluorescence fluctuation spectroscopy (FFS) as a tool for quantitative analysis of protein interactions in cell-free expression systems. We also explore chromophore maturat...

  17. Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70

    OpenAIRE

    Noireaux Vincent; Shin Jonghyeon

    2010-01-01

    Abstract Background Escherichia coli cell-free expression systems use bacteriophage RNA polymerases, such as T7, to synthesize large amounts of recombinant proteins. These systems are used for many applications in biotechnology, such as proteomics. Recently, informational processes have been reconstituted in vitro with cell-free systems. These synthetic approaches, however, have been seriously limited by a lack of transcription modularity. The current available cell-free systems have been opt...

  18. Content of intrinsic disorder influences the outcome of cell-free protein synthesis

    OpenAIRE

    Tokmakov, Alexander A.; Kurotani, Atsushi; Ikeda, Mariko; Terazawa, Yumiko; Shirouzu, Mikako; Stefanov, Vasily; SAKURAI, Tetsuya; Yokoyama, Shigeyuki

    2015-01-01

    Cell-free protein synthesis is used to produce proteins with various structural traits. Recent bioinformatics analyses indicate that more than half of eukaryotic proteins possess long intrinsically disordered regions. However, no systematic study concerning the connection between intrinsic disorder and expression success of cell-free protein synthesis has been presented until now. To address this issue, we examined correlations of the experimentally observed cell-free protein expression yield...

  19. Novel Method of Cell-Free In Vitro Synthesis of the Human Fibroblast Growth Factor 1 Gene

    Directory of Open Access Journals (Sweden)

    Peijun Zuo

    2010-01-01

    Full Text Available Recombinant DNA projects generally involve cell-based gene cloning. However, because template DNA is not always readily available, in vitro chemical synthesis of complete genes from DNA oligonucleotides is becoming the preferred method for cloning. This article describes a new, rapid procedure based on Taq polymerase for the precise assembly of DNA oligonucleotides to yield the complete human fibroblast growth factor 1 (FGF1 gene, which is 468 bp long and has a G+C content of 51.5%. The new method involved two steps: (1 the design of the DNA oligonucleotides to be assembled and (2 the assembly of multiple oligonucleotides by PCR to generate the whole FGF1 gene. The procedure lasted a total of only 2 days, compared with 2 weeks for the conventional procedure. This method of gene synthesis is expected to facilitate various kinds of complex genetic engineering projects that require rapid gene amplification, such as cell-free whole-DNA library construction, as well as the construction of new genes or genes that contain any mutation, restriction site, or DNA tag.

  20. Fetal tachycardia : diagnosis and treatment

    NARCIS (Netherlands)

    Oudijk, Martijn Alexander

    2003-01-01

    Part I: Fetal tachyarrhythmias Diagnosis Fetal tachycardia is a serious condition warranting specialized evaluation. In chapter 2, methods of diagnosis of fetal tachycardia are described, including doppler and M-mode echocardiography and fetal magnetocardiography. The study presented in chapter 3 d

  1. Fetal MR Urography

    OpenAIRE

    Beigi, R.; Abdolmohammad Kajbafzadeh; Alireza Zahiri

    2010-01-01

    Background/Objective: To evaulate the application of fetal MRU in congenital fetal urinary abnormality. The accuracy and appropriate indication of this novel modality are deterimined as well. "nPatients and Methods: Fifty-two mothers were evaluated in an approximate 4.5 year period for genito-urinary (GU) fetal abnormalities. Nearly all patients underwent sonography and MRI performed with signa contour G.E MRI. I will discuss the method and sequence of MRI in detail. "nResults: Feta...

  2. Fetal Alcohol Spectrum Disorders

    Science.gov (United States)

    ... can cause a group of conditions called fetal alcohol spectrum disorders (FASDs). Effects can include physical and behavioral problems such as trouble with Learning and remembering Understanding and following directions Controlling emotions Communicating and socializing Daily life skills, such as ...

  3. Fetal alcohol syndrome

    Science.gov (United States)

    Alcohol in pregnancy; Alcohol-related birth defects; Fetal alcohol effects; FAS ... the baby is in the womb and after birth Decreased muscle tone and ... Heart defects such as ventricular septal defect (VSD) or atrial ...

  4. Fetal MR Urography

    Directory of Open Access Journals (Sweden)

    R. Beigi

    2010-05-01

    Full Text Available Background/Objective: To evaulate the application of fetal MRU in congenital fetal urinary abnormality. The accuracy and appropriate indication of this novel modality are deterimined as well. "nPatients and Methods: Fifty-two mothers were evaluated in an approximate 4.5 year period for genito-urinary (GU fetal abnormalities. Nearly all patients underwent sonography and MRI performed with signa contour G.E MRI. I will discuss the method and sequence of MRI in detail. "nResults: Fetal MRU was the sensitive method in detecting most GU abnormalities, such as ureteropelvic junction (UPJ stenosis or posterior urethral valve (PUV or mega ureter. "nConclusion: This method is not as skill dependent as sonography and it permits whole evaluation of the GU system and facilated preoprative planning.    

  5. Fetal and neonatal thyrotoxicosis

    Directory of Open Access Journals (Sweden)

    Chandar Mohan Batra

    2013-01-01

    Full Text Available Fetal thyrotoxicosis is a rare disease occurring in 1 out of 70 pregnancies with Grave′s disease or in 1 out of 4000-50,000 deliveries. The mortality is 12-20%, usually from heart failure, but other complications are tracheal compression, infections and thrombocytopenia. It results from transfer of thyroid stimulating immunoglobulins from mother to fetus through the placenta. This transplacental transfer begins around 20 th week of pregnancy and reaches its maximum by 30 th week. These autoantibodies bind to the fetal thyroid stimulating hormone (TSH receptors and increase the secretion of the thyroid hormones. The mother has an active autoimmune thyroid disease or has been treated for it in the past. She may be absolutely euthyroid due to past treatment by drugs, surgery or radioiodine ablation, but still have active TSH receptor stimulating autoantibodies, which can cause fetal thyrotoxicosis. The other features of this disease are fetal tachycardia, fetal goiter and history of spontaneous abortions and findings of goiter, ascites, craniosyntosis, fetal growth retardation, maceration and hydrops at fetal autopsy. If untreated, this disease can result in intrauterine death. The treatment for this disease consists of giving carbimazole to the mother, which is transferred through the placenta to the fetus. The dose of carbimazole is titrated with the fetal heart rate. If the mother becomes hypothyroid due to carbimazole, thyroxine is added taking advantage of the fact that very little of thyroxine is transferred across the placenta. Neonatal thyrotoxicosis patients are very sick and require emergency treatment. The goal of the treatment is to normalize thyroid functions as quickly as possible, to avoid iatrogenic hypothyroidism while providing management and supportive therapy for the infant′s specific signs and symptoms.

  6. Expression of Green Fluorescent Protein (GFP using In Vitro translation cell free system

    Directory of Open Access Journals (Sweden)

    M Mohamadipoor

    2009-03-01

    Full Text Available ABSTRACT Background and the purpose of the study: One of the major concerns about recombinant protein production is its possible toxicity for the organism. Purification of the recombinant protein is another challenge in this respect. Recently In Vitro translation cell free system that provides a coupled transcription-translation reaction for protein synthesis to overcome the above mentioned problems has been emerged. The aim of this study was expression of GFP as a marker for gene expression and protein in In Vitro translation system. Methods: pIVEX2.3-GFP plasmid was cloned to E. coli   and the plasmid DNA extracted. In Vitro translation was performed with RTS 100 E. coli Hy kit according to manufacture's instructions. Expression of recombinant fusion protein, His- GFP, was determined by SDS-PAGE, ELISA and western blot analysis. Results: Expected size of recombinant protein was detected in SDS-PAGE and further confirmed by western blot analysis and ELISA. Major conclusion: Results showed that In Vitro translation is suitable for expression of recombinant protein and fusion of the recombinant protein with His-tag facilitates the purification.

  7. Liquid biopsy of gastric cancer patients: circulating tumor cells and cell-free nucleic acids.

    Science.gov (United States)

    Tsujiura, Masahiro; Ichikawa, Daisuke; Konishi, Hirotaka; Komatsu, Shuhei; Shiozaki, Atsushi; Otsuji, Eigo

    2014-03-28

    To improve the clinical outcomes of cancer patients, early detection and accurate monitoring of diseases are necessary. Numerous genetic and epigenetic alterations contribute to oncogenesis and cancer progression, and analyses of these changes have been increasingly utilized for diagnostic, prognostic and therapeutic purposes in malignant diseases including gastric cancer (GC). Surgical and/or biopsy specimens are generally used to understand the tumor-associated alterations; however, those approaches cannot always be performed because of their invasive characteristics and may fail to reflect current tumor dynamics and drug sensitivities, which may change during the therapeutic process. Therefore, the importance of developing a non-invasive biomarker with the ability to monitor real-time tumor dynamics should be emphasized. This concept, so called "liquid biopsy", would provide an ideal therapeutic strategy for an individual cancer patient and would facilitate the development of "tailor-made" cancer management programs. In the blood of cancer patients, the presence and potent utilities of circulating tumor cells (CTCs) and cell-free nucleic acids (cfNAs) such as DNA, mRNA and microRNA have been recognized, and their clinical relevance is attracting considerable attention. In this review, we discuss recent developments in this research field as well as the relevance and future perspectives of CTCs and cfNAs in cancer patients, especially focusing on GC. PMID:24696609

  8. Maternal aldehyde elimination during pregnancy preserves the fetal genome.

    Science.gov (United States)

    Oberbeck, Nina; Langevin, Frédéric; King, Gareth; de Wind, Niels; Crossan, Gerry P; Patel, Ketan J

    2014-09-18

    Maternal metabolism provides essential nutrients to enable embryonic development. However, both mother and embryo produce reactive metabolites that can damage DNA. Here we discover how the embryo is protected from these genotoxins. Pregnant mice lacking Aldh2, a key enzyme that detoxifies reactive aldehydes, cannot support the development of embryos lacking the Fanconi anemia DNA repair pathway gene Fanca. Remarkably, transferring Aldh2(-/-)Fanca(-/-) embryos into wild-type mothers suppresses developmental defects and rescues embryonic lethality. These rescued neonates have severely depleted hematopoietic stem and progenitor cells, indicating that despite intact maternal aldehyde catabolism, fetal Aldh2 is essential for hematopoiesis. Hence, maternal and fetal aldehyde detoxification protects the developing embryo from DNA damage. Failure of this genome preservation mechanism might explain why birth defects and bone marrow failure occur in Fanconi anemia, and may have implications for fetal well-being in the many women in Southeast Asia that are genetically deficient in ALDH2. PMID:25155611

  9. Cell-free protein synthesis of a cytotoxic cancer therapeutic: Onconase production and a just-add-water cell-free system.

    Science.gov (United States)

    Salehi, Amin S M; Smith, Mark Thomas; Bennett, Anthony M; Williams, Jacob B; Pitt, William G; Bundy, Bradley C

    2016-02-01

    Biotherapeutics have many promising applications, such as anti-cancer treatments, immune suppression, and vaccines. However, due to their biological nature, some biotherapeutics can be challenging to rapidly express and screen for activity through traditional recombinant methods. For example, difficult-to-express proteins may be cytotoxic or form inclusion bodies during expression, increasing the time, labor, and difficulty of purification and downstream characterization. One potential pathway to simplify the expression and screening of such therapeutics is to utilize cell-free protein synthesis. Cell-free systems offer a compelling alternative to in vivo production, due to their open and malleable reaction environments. In this work, we demonstrate the use of cell-free systems for the expression and direct screening of the difficult-to-express cytotoxic protein onconase. Using cell-free systems, onconase can be rapidly expressed in soluble, active form. Furthermore, the open nature of the reaction environment allows for direct and immediate downstream characterization without the need of purification. Also, we report the ability of a "just-add-water" lyophilized cell-fee system to produce onconase. This lyophilized system remains viable after being stored above freezing for up to one year. The beneficial features of these cell-free systems make them compelling candidates for future biotherapeutic screening and production. PMID:26380966

  10. Maternal-fetal thyroid hormone relationships and the fetal brain.

    Science.gov (United States)

    Morreale de Escobar, G; Obregon, M J; Escobar del Rey, F

    1988-01-01

    Thyroid hormones are transferred from the mother to the fetus. Thus, despite the deiodinating enzymes of the placenta (26), some T4 and T3 is transferred, both before and after onset of fetal thyroid function, at least in those cases where fetal thyroid function is impaired. It is also possible that transfer occurs under normal conditions. Maternal to fetal transfer of T3 and T4 is partially limited. But it might be enough to mitigate severe fetal T4 and T3 deficiencies. However, the mitigating effects of both hormones are not equivalent for all fetal tissues. 1) Maternal T4 mitigates T4 and T3 deficiency of most fetal tissues, the brain included. 2) Maternal T3 mitigates T3 deficiency only in some fetal tissues, the brain being excluded. It does not mitigate cerebral T3 deficiency even at doses which are toxic for the mother, and it does not depress fetal plasma TSH. 3) Normal maternal thyroid function is important for fetal development. Maternal hypothyroxinemia is damaging to the developing fetal brain early in gestation. It might also later have adverse effects in gestation, if the fetal thyroid is impaired. Normal maternal T3 levels might avoid overt hypothyroidism of some fetal tissues, but is of no benefit to the brain. PMID:3176827

  11. Fetal abdominal magnetic resonance imaging

    International Nuclear Information System (INIS)

    This review deals with the in vivo magnetic resonance imaging (MRI) appearance of the human fetal abdomen. Imaging findings are correlated with current knowledge of human fetal anatomy and physiology, which are crucial to understand and interpret fetal abdominal MRI scans. As fetal MRI covers a period of more than 20 weeks, which is characterized not only by organ growth, but also by changes and maturation of organ function, a different MR appearance of the fetal abdomen results. This not only applies to the fetal intestines, but also to the fetal liver, spleen, and adrenal glands. Choosing the appropriate sequences, various aspects of age-related and organ-specific function can be visualized with fetal MRI, as these are mirrored by changes in signal intensities. Knowledge of normal development is essential to delineate normal from pathological findings in the respective developmental stages

  12. Fetal abdominal magnetic resonance imaging

    Energy Technology Data Exchange (ETDEWEB)

    Brugger, Peter C. [Center of Anatomy and Cell Biology, Integrative Morphology Group, Medical University of Vienna, Waehringerstrasse 13, 1090 Vienna (Austria)]. E-mail: peter.brugger@meduniwien.ac.at; Prayer, Daniela [Department of Radiology, Medical University of Vienna, Waehringerguertel 18-20, 1090 Vienna (Austria)

    2006-02-15

    This review deals with the in vivo magnetic resonance imaging (MRI) appearance of the human fetal abdomen. Imaging findings are correlated with current knowledge of human fetal anatomy and physiology, which are crucial to understand and interpret fetal abdominal MRI scans. As fetal MRI covers a period of more than 20 weeks, which is characterized not only by organ growth, but also by changes and maturation of organ function, a different MR appearance of the fetal abdomen results. This not only applies to the fetal intestines, but also to the fetal liver, spleen, and adrenal glands. Choosing the appropriate sequences, various aspects of age-related and organ-specific function can be visualized with fetal MRI, as these are mirrored by changes in signal intensities. Knowledge of normal development is essential to delineate normal from pathological findings in the respective developmental stages.

  13. Expression optimization and synthetic gene networks in cell-free systems

    OpenAIRE

    Karig, David K.; Iyer, Sukanya; Simpson, Michael L.; Doktycz, Mitchel J.

    2011-01-01

    Synthetic biology offers great promise to a variety of applications through the forward engineering of biological function. Most efforts in this field have focused on employing living cells, yet cell-free approaches offer simpler and more flexible contexts. Here, we evaluate cell-free regulatory systems based on T7 promoter-driven expression by characterizing variants of TetR and LacI repressible T7 promoters in a cell-free context and examining sequence elements that determine expression eff...

  14. Effect of erythrocyte aggregation and flow rate on cell-free layer formation in arterioles

    OpenAIRE

    Ong, Peng Kai; Namgung, Bumseok; Johnson, Paul C.; Kim, Sangho

    2010-01-01

    Formation of a cell-free layer is an important dynamic feature of microcirculatory blood flow, which can be influenced by rheological parameters, such as red blood cell aggregation and flow rate. In this study, we investigate the effect of these two rheological parameters on cell-free layer characteristics in the arterioles (20–60 μm inner diameter). For the first time, we provide here the detailed temporal information of the arteriolar cell-free layer in various rheological conditions to bet...

  15. Fetal Biophysical Profile Scoring

    Directory of Open Access Journals (Sweden)

    H.R. HaghighatKhah

    2009-01-01

    Full Text Available   "nFetal biophysical profile scoring is a sonographic-based method of fetal assessment first described by Manning and Platt in 1980. "nThe biophysical profile score was developed as a method to integrate real-time observations of the fetus and his/her intrauterine environment in order to more comprehensively assess the fetal condition. These findings must be evaluated in the context of maternal/fetal history (i.e., chronic hypertension, post-dates, intrauterine growth restriction, etc, fetal structural integrity (presence or absence of congenital anomalies, and the functionality of fetal support structures (placental and umbilical cord. For example, acute asphyxia due to placental abruption may result in an absence of the acute variables of the biophysical profile score (fetal breathing movements, fetal movement, fetal tone, and fetal heart rate reactivity with a normal amniotic fluid volume. With post maturity the asphyxial event may be intermittent and chronic resulting in a decrease in amniotic fluid volume, but with the acute variables remaining normal. "nWhile the 5 components of the biophysical profile score have remained unchanged since 1980 (Manning, 1980, the definitions of a normal and abnormal parameter have evolved with increasing experience. "nIn 1984 the definition of oligohydramnios was increased from < 1cm pocket of fluid to < 2.0 x 1.0 cm pocket. Oligohydramnios is now defined as a pocket of amniotic fluid < 2.0 x 2.0 cm (Manning, 1995a "nIf the four ultrasound variables are normal, the accuracy of the biophysical profile score was not found to be significantly improved by adding the non-stress test. As a result, in 1987 the profile score was modified to incorporate the non-stress test only when one of the ultrasound variables was abnormal (Manning 1987. Table 1 outlines the current definitions for quantifying a variable as present or absent. "nEach of the 5 components of the biophysical profile score does not have equal

  16. Fetal fluid and protein dynamics

    OpenAIRE

    Pasman, Suzanne

    2010-01-01

    In this thesis fetal fluid and protein dynamics are investigated to gain insight in fetal (patho-)physiology. Studies were performed in fetuses with severe anemia and/or hydrops fetalis. Measurements were performed in fetal blood or amniotic fluid, obtained before or during intrauterine transfusion. The severity of anemia can be predicted by measurement of bilirubin in amniotic fluid. We showed that this concentration is based on bilirubin in fetal blood and on albumin concentrations. Albumin...

  17. CELLULAR FETAL MICROCHIMERISM IN PREECLAMPSIA

    OpenAIRE

    Gammill, Hilary S; Aydelotte, Tessa M.; Guthrie, Katherine A.; Nkwopara, Evangelyn C.; Nelson, J. Lee

    2013-01-01

    Previous studies have shown elevated concentrations of free fetal deoxyribonucleic acid and erythroblasts in maternal circulation in preeclampsia compared with normal pregnancy. Pluripotent and immunocompetent fetal cells also transfer to the maternal circulation during pregnancy, but whether concentrations of fetal mononuclear cells also differed in preeclampsia was unknown. We sought to quantify cellular fetal microchimerism in maternal circulation in women with preeclampsia and healthy con...

  18. Fetal Amelia: A Case Report

    OpenAIRE

    Mohammed Abdul-Latif; Asfhaq Ahmed; Shahila Tanzeem; Nihal Al Riyami

    2012-01-01

     Congenital limb defects are rare fetal anomalies with a birth prevalence of 0.55 per 1,000. Amelia is an extremely rare birth defect marked by the complete absence of one or more limbs. We report a case of fetal amelia, ultrasound findings, manifestations and the fetal outcome.

  19. Characterization of the Cell-Free Layer in a Microvessel by Computer Simulation

    Science.gov (United States)

    Jee, Sol Keun; Freund, Jonathon; Moser, Robert

    2006-11-01

    The cell-free layer between the erythrocyte-rich core of a micro-vessel and the vessel wall is a significant component of the hydrodynamics of the microcirculation. To investigate the mechanics of the cell-free layer, we simulate a two-dimensional periodic blood flow in a microvessel containing numerous erythrocytes, modeled as capsules with elastic shell membranes using the boundary integral method. Cell-cell interactions are mediated with an interaction potential which represents aggregation forces. Our model successfully recreates in-vivo hemodynamic properties such as blunt velocity profile and Fahraeus effect. The cell-free layer has a thickness of order one erythrocyte radius which is consistent with experimental results. To investigate the mechanics of the cell-free layer a number of numerical experiments were conducted, in which the effects of aggregation forces, and lubrication forces are investigated, by varying the aggregation potential, introducing artificial body forces and changing boundary condition.

  20. Fetal Alcohol Syndrome.

    Science.gov (United States)

    Zerrer, Peggy

    The paper reviews Fetal Alcohol Syndrome (FAS), a series of effects seen in children whose mothers drink alcohol to excess during pregnancy. The identification of FAS and its recognition as a major health problem in need of prevention are traced. Characteristics of children with FAS are described and resultant growth retardation, abnormal physical…

  1. Cell-Free Expression of Protein Kinase A for Rapid Activity Assays

    OpenAIRE

    Leippe, Donna M.; Kate Qin Zhao; Kevin Hsiao; Slater, Michael R.

    2010-01-01

    Functional protein analysis often calls for lengthy, laborious in vivo protein expression and purification, and can be complicated by the lack of stability of the purified protein. In this study, we demonstrate the feasibility of a simplified procedure for functional protein analysis on magnetic particles using cell-free protein synthesis of the catalytic subunit of human cAMP-dependent protein kinase as a HaloTag® fusion protein. The cell-free protein synthesis systems provide quick access t...

  2. Cell-free biology: exploiting the interface between synthetic biology and synthetic chemistry

    OpenAIRE

    Harris, D. Calvin; Jewett, Michael C.

    2012-01-01

    Just as synthetic organic chemistry once revolutionized the ability of chemists to build molecules (including those that did not exist in nature) following a basic set of design rules, cell-free synthetic biology is beginning to provide an improved toolbox and faster process for not only harnessing but also expanding the chemistry of life. At the interface between chemistry and biology, research in cell-free synthetic systems is proceeding in two different directions: using synthetic biology ...

  3. Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70

    Directory of Open Access Journals (Sweden)

    Noireaux Vincent

    2010-06-01

    Full Text Available Abstract Background Escherichia coli cell-free expression systems use bacteriophage RNA polymerases, such as T7, to synthesize large amounts of recombinant proteins. These systems are used for many applications in biotechnology, such as proteomics. Recently, informational processes have been reconstituted in vitro with cell-free systems. These synthetic approaches, however, have been seriously limited by a lack of transcription modularity. The current available cell-free systems have been optimized to work with bacteriophage RNA polymerases, which put significant restrictions to engineer processes related to biological information. The development of efficient cell-free systems with broader transcription capabilities is required to study complex informational processes in vitro. Results In this work, an efficient cell-free expression system that uses the endogenous E. coli RNA polymerase only and sigma factor 70 for transcription was prepared. Approximately 0.75 mg/ml of Firefly luciferase and enhanced green fluorescent protein were produced in batch mode. A plasmid was optimized with different regulatory parts to increase the expression. In addition, a new eGFP was engineered that is more translatable in cell-free systems than the original eGFP. The protein production was characterized with three different adenosine triphosphate (ATP regeneration systems: creatine phosphate (CP, phosphoenolpyruvate (PEP, and 3-phosphoglyceric acid (3-PGA. The maximum protein production was obtained with 3-PGA. Preparation of the crude extract was streamlined to a simple routine procedure that takes 12 hours including cell culture. Conclusions Although it uses the endogenous E. coli transcription machinery, this cell-free system can produce active proteins in quantities comparable to bacteriophage systems. The E. coli transcription provides much more possibilities to engineer informational processes in vitro. Many E. coli promoters/operators specific to sigma

  4. Using fetal cells for prenatal diagnosis: History and recent progress.

    Science.gov (United States)

    Beaudet, Arthur L

    2016-06-01

    The potential to use fetal cells in the mother's circulation during the first or second trimester for prenatal diagnosis was described in 1968, but it has not been possible do develop a routine clinical prenatal test despite extensive commercial and academic research efforts. Early attention focused on the detection of aneuploidy, but more recent technology opens the possibility of high resolution detection of copy number abnormalities and even whole genome or exome sequencing to detect both inherited and de novo mutations. In the interim, cell-free noninvasive prenatal testing NIPT has allowed improved detection of aneuploidy, but this has led to a sharp reduction in the number of amniocentesis and chorionic villus sampling (CVS) procedures, which inevitably implies reduced detection of serious de novo deletion abnormalities. Attention has focused of both fetal nucleated red blood cells (fnRBCs) and trophoblasts. Recent progress presented at meetings, but not yet published, suggests that it will soon be possible to perform genome-wide relatively high resolution detection of deletions and duplications by recovering fetal trophoblasts during the first trimester and analyzing them by whole gene genome amplification followed by copy number analysis using arrays or next generation sequencing. © 2016 Wiley Periodicals, Inc. PMID:27133782

  5. Prognostic importance of cell-free DNA in chemotherapy resistant ovarian cancer treated with bevacizumab

    DEFF Research Database (Denmark)

    Steffensen, Karina Dahl; Madsen, Christine Vestergaard; Andersen, Rikke Fredslund;

    2014-01-01

    AIM: Treatment of multiresistant epithelial ovarian cancer (EOC) is palliative and patients who have become resistant after multiple lines of chemotherapy often have an unmet need for further and less toxic treatment. Anti-angiogenic therapy has attracted considerable attention in the treatment of...... EOC in combination with chemotherapy. However, only a minor subgroup will benefit from the treatment and there is an obvious need for new markers to select such patients. The purpose of this study was to investigate the effect of single-agent bevacizumab in multiresistant EOC and the importance of......). RESULTS: Eighteen percent responded to treatment according to CA125 and 5.6% had partial response by Response Evaluation Criteria in Solid Tumours (RECIST). Stable disease was seen in 53.5% and 48.6% of the patients by CA125 and RECIST, respectively. Median progression free survival (PFS) and overall...

  6. Tumour pharmacodynamics and circulating cell free DNA in patients with refractory colorectal carcinoma treated with regorafenib

    OpenAIRE

    Wong, Andrea Li Ann; Lim, Joline Si Jing; Sinha, Arvind; Gopinathan, Anil; Lim, Robert; Tan, Chee-Seng; Soh, Thomas; Venkatesh, Sudhakar; Titin, Christina; Sapari, Nur Sabrina; Lee, Soo-Chin; Yong, Wei-Peng; Tan, David Shao Ping; Pang, Brendan; Wang, Ting-Ting

    2015-01-01

    Background Regorafenib, a multi-kinase inhibitor, is used in the treatment of patients with metastatic colorectal cancer refractory to standard therapy. However, this benefit was limited to 1.4 months improvement in overall survival, with more than half of patients experiencing grade 3 to 4 adverse events. We aim to elucidate the pharmacodynamic effects of regorafenib in metastatic colorectal cancer and discover potential biomarkers that may predict clinical benefit. Methods Patients with met...

  7. Interaction of DNA with anticancer ruthenium complexes containing dimethylsulfoxide ligands in cell-free media

    Czech Academy of Sciences Publication Activity Database

    Nováková, Olga; Alessio, E.; Brabec, Viktor

    Seville, 1998, s. 286. [Fourth European Biological Inorganic Chemistry Conference. Seville (ES), 20.07.1998-25.07.1998] Institutional research plan: CEZ:A17/98:Z5-004-9-ii Subject RIV: BO - Biophysics

  8. Microarray-Based Differential Expression Monitoring of 79 Novel Genes in Human Fetal Tissues

    Institute of Scientific and Technical Information of China (English)

    Ma; Shu-hua; Wang; Dun-cheng; 等

    2003-01-01

    79 ESTs fragments with represents corresponding novel genes were obtained by sequencing and bioinformatics analysis of human fetal kidney cDNA library. Microarray was prepared by using these novel EST fragments by automatic spotting. Expression patters of 79 ESTs of novel genes from human fetal kidney were analyzed in fetal brain and fetal heart tissues of 20-week-and 26-week-age fetus by performing of cDNA chip hybridization. This provides clues for studying exact functions of the novel genes. 8 genes were obtained which were expressed differentially in the fetal brain and heart of 20-week-and 26-week-age respectively. Then differentially expressed genes were identified by Northern analysis. The more exact function of the novel genes is under study.

  9. Fetal varicella syndrome

    Directory of Open Access Journals (Sweden)

    Ramachandra S

    2010-01-01

    Full Text Available Fetal varicella syndrome is a rare condition of the newborn, presenting with cutaneous scars, limb defects and ocular and central nervous system abnormalities. It is due to varicella or zoster developing in the fetus following maternal varicella infection during early pregnancy. We are reporting one such patient who presented with a linear, depressed, erythematous scar over the left forearm and axillary fold, with a history of maternal chicken pox during the first trimester of pregnancy.

  10. Fetal varicella syndrome

    OpenAIRE

    Ramachandra S; Metta Arun; Haneef Nayeem; Kodali Sandeep

    2010-01-01

    Fetal varicella syndrome is a rare condition of the newborn, presenting with cutaneous scars, limb defects and ocular and central nervous system abnormalities. It is due to varicella or zoster developing in the fetus following maternal varicella infection during early pregnancy. We are reporting one such patient who presented with a linear, depressed, erythematous scar over the left forearm and axillary fold, with a history of maternal chicken pox during the first trimester of pregnancy.

  11. Maternal-fetal conflict.

    Science.gov (United States)

    Fasouliotis, S J; Schenker, J G

    2000-03-01

    Advances in prenatal care have brought about a greater understanding as to the special status of the fetus to the point that it is considered a patient in its own regard. Pregnant women generally follow the medical recommendations of their physicians that are intended for the benefit of their baby. Any situation where maternal well-being or wishes contradict fetal benefit constitutes a maternal-fetal conflict. Such situations include a broad range of possible interventions, non-interventions, and coercive influences. In such cases, the attending physician is expected to attain an attitude that involves either the respect of the woman's autonomy and right to privacy, which precludes any approach other than to accept her decision, or to modify this absolute for the beneficence of the fetus. Current ethical viewpoints range from absolute respect for maternal autonomy with no persuasion allowed, to gentle persuasion and to others which permit intervention and overriding of the woman's autonomy. Court-ordered decisions enforcing the pregnant woman to undergo a procedure in order to improve fetal outcome have been criticized as an invasion of a woman's privacy, limitation of her autonomy, and taking away of her right to informed consent. PMID:10733034

  12. Fetal magnetic resonance: technique applications and normal fetal anatomy

    International Nuclear Information System (INIS)

    Ultrasonography is the preferred diagnostic imaging technique for intrauterine fetal examination. Nevertheless, circumstances sometimes dictate the use of other techniques in order to analyze fetal structures. The advent of ultra rapid magnetic resonance (MR) sequencing has led to the possibility of doing MR fetal studies, since images are obtained in an extradordiarily short time and are not affected by either maternal or fetal movements. It does not employ ionizing radiations, it provides high-contrast images and it can obtain such images in any plane of space without being influenced by either the child bearer's physical characteristics of fetal position. MR provides good quality images of most fetal organs. It is extremely useful in analysing distinct structures, as well as permitting an evaluation of cervical structures, lungs, diaphragms, intra-abdominal and retroperitoneal structures, and fetal extremities. It can also provide useful information regarding the placenta,umbilical cord, amniotic fluid and uterus. The objective of this work is to describe MR technique as applied to intrauterine fetal examination, and to illustrate normal fetal anatomy as manifested by MR and its applications. (Author) 42 refs

  13. Management of fetal growth restriction

    OpenAIRE

    Alberry, M; Soothill, P

    2007-01-01

    Fetal growth restriction (FGR) is challenging because of the difficulties in reaching a definitive diagnosis of the cause and planning management. FGR is associated not only with a marked increased risk in perinatal mortality and morbidity but also with long‐term outcome risks. Combinations of fetal biometry, amniotic fluid volume, heart rate patterns, arterial and venous Doppler, and biophysical variables allow a comprehensive fetal evaluation of FGR. However, no evidence supports that the u...

  14. Cell-free microRNAs in blood and other body fluids, as cancer biomarkers.

    Science.gov (United States)

    Ortiz-Quintero, Blanca

    2016-06-01

    The discovery of cell-free microRNAs (miRNAs) in serum, plasma and other body fluids has yielded an invaluable potential source of non-invasive biomarkers for cancer and other non-malignant diseases. miRNAs in the blood and other body fluids are highly stable in biological samples and are resistant to environmental conditions, such as freezing, thawing or enzymatic degradation, which makes them convenient as potential biomarkers. In addition, they are more easily sampled than tissue miRNAs. Altered levels of cell-free miRNAs have been found in every type of cancer analysed, and increasing evidence indicates that they may participate in carcinogenesis by acting as cell-to-cell signalling molecules. This review summarizes the biological characteristics and mechanisms of release of cell-free miRNAs that make them promising candidates as non-invasive biomarkers of cancer. PMID:27218664

  15. Atomic force microscopy observation on nuclear reassembly in a cell-free system

    Institute of Scientific and Technical Information of China (English)

    YANG Ning; CHEN Zhongcai; ZHANG Zhaohui; ZHU Xing; ZHAI Zhonghe; TANG Xiaowei

    2003-01-01

    Cell-free system is interesting and useful for studying nuclear assembly in mitosis. Atomic force micro- scopy (AFM), which is a simple way for imaging fixed reassemble nuclei with high resolution, has not been used in the cell-free system. In this paper, we put forward an air-drying sample preparation for AFM. Using AFM, we observed nuclear reassembly process within 100 nm resolution ina cell-free system. As a result, we found that the images were artifact-free, and with higher resolution compared with fluorescent optical microscope images. Furthermore, the morphology of membrane vesicles was obtained clearly, and a dynamic change of morphology during the vesicles' approaching to nuclear envelope was also observed, which is enlightened to understand the mechanism of nuclear envelope assembly.

  16. Fetal Alcohol Spectrum Disorders (FASDs): Diagnosis

    Science.gov (United States)

    ... through Biomarkers International Research National Task Force on Fetal Alcohol Syndrome and Fetal Alcohol Effect Training & Education Past Activities ... review the research and develop guidelines for diagnosing fetal alcohol syndrome (FAS) . The guidelines were developed for FAS only. ...

  17. Fetal Alcohol Syndrome and Fetal Alcohol Effects in Child Development.

    Science.gov (United States)

    Pancratz, Diane R.

    This literature review defines Fetal Alcohol Syndrome (FAS) and Fetal Alcohol Effects (FAE) and considers their causes, diagnoses, prevalence, and educational ramifications. Effects of alcohol during each of the trimesters of pregnancy are summarized. Specific diagnostic characteristics of FAS are listed: (1) growth deficiency, (2) a…

  18. Assessment of fetal heart disorder by means of fetal magnetocardiography

    Science.gov (United States)

    Łozińska, Maria; Dunajski, Zbigniew

    2006-10-01

    Fetal magnetocardiography is new method for investigations of electrical activity of the fetal heart. The idea and build of system for magnetic signal registration is described. Two cases of premature atrial contraction and complete AV block diagnosis by means of magnetic field recording system are described.

  19. Are there fetal stem cells in the maternal brain?

    Science.gov (United States)

    Demirhan, Osman; Cekin, Necmi; Taştemir, Deniz; Tunç, Erdal; Güzel, Ali İrfan; Meral, Demet; Demirbek, Bülent

    2013-03-01

    Fetal cells can enter maternal blood during pregnancy but whether they can also cross the blood-brain barrier to enter the maternal brain remains poorly understood. Previous results suggest that fetal cells are summoned to repair damage to the mother's brain. If this is confirmed, it would open up new and safer avenues of treatment for brain damage caused by strokes and neural diseases. In this study, we aimed to investigate whether a baby's stem cells can enter the maternal brain during pregnancy. Deceased patients who had at least one male offspring and no history of abortion and blood transfusion were included in this study. DNA was extracted from brain tissue samples of deceased women using standard phenol-chloroform extraction and ethanol precipitation methods. Genomic DNA was screened by quantitative fluorescent-polymerase chain reaction amplification together with short tandem repeat markers specific to the Y chromosome, and 13, 18, 21 and X. Any foreign DNA residues that could be used to interpret the presence of fetal stem cells in the maternal brain were monitored. Results indicated that fetal stem cells can not cross the blood-brain barrier to enter the maternal brain. PMID:25206703

  20. Are there fetal stem cells in the maternal brain?

    Institute of Scientific and Technical Information of China (English)

    Osman Demirhan; Necmi (C)ekin; Deniz Ta(s)temir; Erdal Tun(c); Ali irfan Güzel; Demet Meral; Bülent Demirbek

    2013-01-01

    Fetal cells can enter maternal blood during pregnancy but whether they can also cross the blood-brain barrier to enter the maternal brain remains poorly understood. Previous results suggest that fetal cells are summoned to repair damage to the mother's brain. If this is confirmed, it would open up new and safer avenues of treatment for brain damage caused by strokes and neural diseases. In this study, we aimed to investigate whether a baby's stem cells can enter the maternal brain during pregnancy. Deceased patients who had at least one male offspring and no history of abortion and blood transfusion were included in this study. DNA was extracted from brain tissue samples of deceased women using standard phenol-chloroform extraction and ethanol precipitation methods. Genomic DNA was screened by quantitative fluorescent-polymerase chain reaction amplification together with short tandem repeat markers specific to the Y chromosome, and 13, 18, 21 and X. Any foreign DNA residues that could be used to interpret the presence of fetal stem cells in the maternal brain were monitored. Results indicated that fetal stem cells can not cross the blood-brain barrier to enter the maternal brain.

  1. A cell-free protein synthesis system for high-throughput proteomics

    OpenAIRE

    Sawasaki, Tatsuya; Ogasawara, Tomio; Morishita, Ryo; Endo, Yaeta

    2002-01-01

    We report a cell-free system for the high-throughput synthesis and screening of gene products. The system, based on the eukaryotic translation apparatus of wheat seeds, has significant advantages over other commonly used cell-free expression systems. To maximize the yield and throughput of the system, we optimized the mRNA UTRs, designed an expression vector for large-scale protein production, and developed a new strategy to construct PCR-generated DNAs for high-throughput production of many ...

  2. Comparison between human fetal and adult skin

    OpenAIRE

    Coolen, N.A.; Schouten, K.C.; Middelkoop, E.; Ulrich, M.

    2009-01-01

    Healing of early-gestation fetal wounds results in scarless healing. Since the capacity for regeneration is probably inherent to the fetal skin itself, knowledge of the fetal skin composition may contribute to the understanding of fetal wound healing. The aim of this study was to analyze the expression profiles of different epidermal and dermal components in the human fetal and adult skin. In the human fetal skin (ranging from 13 to 22 weeks’ gestation) and adult skin biopsies, the expression...

  3. Ovine fetal necrobacillosis

    DEFF Research Database (Denmark)

    Agerholm, J.S.; Boye, Mette; Aalbæk, B.

    2007-01-01

    were found in several tissues. Histologically, placental lesions were characterized by locally diffuse infiltration of neutrophils, closely associated with abundant small Gram-negative and FISH-positive rods, thrombosis and necrosis. Lesions in the fetal-maternal interface were multifocal and consisted...... spread and infection of the fetus through the amniotic fluid was characterized by development of multifocal suppurative dermatitis and suppurative bronchopneumonia. Localization of FISH-positive bacteria in necrotic lesions was restricted to the periphery. E necrophorum would seem to have been...

  4. Restrictive dermopathy and fetal behaviour

    NARCIS (Netherlands)

    Mulder, EJH; Beemer, FA; Stoutenbeek, P

    2001-01-01

    We report three siblings from consecutive pregnancies affected with restrictive dermopathy (RD). During the second pregnancy, fetal behavioural development and growth were studied extensively using ultrasound at 1-4 week intervals. Dramatic and sudden changes occurred in fetal body movements and gro

  5. Impact of fetal echocardiography

    International Nuclear Information System (INIS)

    Prenatal diagnosis of congenital heart disease is now well established for a wide range of cardiac anomalies. Diagnosis of congenital heart disease during fetal life not only identifies the cardiac lesion but may also lead to detection of associated abnormalities. This information allows a detailed discussion of the prognosis with parents. For continuing pregnancies, appropriate preparation can be made to optimize the postnatal outcome. Reduced morbidity and mortality, following antenatal diagnosis, has been reported for coarctation of the aorta, hypoplastic left heart syndrome, and transposition of the great arteries. With regard to screening policy, most affected fetuses are in the “low risk” population, emphasizing the importance of appropriate training for those who undertake such obstetric anomaly scans. As a minimum, the four chamber view of the fetal heart should be incorporated into midtrimester anomaly scans, and where feasible, views of the outflow tracts should also be included, to increase the diagnostic yield. Newer screening techniques, such as measurement of nuchal translucency, may contribute to identification of fetuses at high risk for congenital heart disease and prompt referral for detailed cardiac assessment

  6. Cell-free plasma microRNA in pancreatic ductal adenocarcinoma and disease controls

    DEFF Research Database (Denmark)

    Carlsen, Anting Liu; Joergensen, Maiken Thyregod; Knudsen, Steen;

    2013-01-01

    There are no tumor-specific biochemical markers for pancreatic ductal adenocarcinoma (PDAC). Tissue-specific gene expression including microRNA (miRNA) profiling, however, identifies specific PDAC signatures. This study evaluates associations between circulating, cell-free plasma-miRNA profiles and...... PDAC in a disease and disease-control cohort....

  7. Fetal and maternal outcomes in pregnancies complicated with fetal macrosomia

    Directory of Open Access Journals (Sweden)

    Mohamed Alkhatim Alsammani

    2012-01-01

    Full Text Available Background: Fetal macrosomia remains a considerable challenge in current obstetrics due to the fetal and maternal complications associated with this condition. Aim: This study was designed to determine the prevalence of fetal macrosomia and associated fetal and maternal morbidity and mortality in the Al Qassim Region of Saudi Arabia. Materials and Methods: This register-based study was conducted from January 1, 2011 through December 30, 2011 at the Maternity and Child Hospital, Qassim, Saudi Arabia. Macrosomia was defined as birth weight of 4 kg or greater. Malformed babies and those born dead were excluded. Results: The total number of babies delivered was 9241; of these, 418 were macrosomic. Thus, the prevalence of fetal macrosomia was 4.5%. The most common maternal complications were postpartum hemorrhage (5 cases, 1.2%, perineal tear (7 cases, 1.7%, cervical lacerations (3 cases, 0.7%, and shoulder dystocia (40 cases, 9.6% that resulted in 4 cases of Erb′s palsy (0.96%, and 6 cases of bone fractures (1.4%. The rate of cesarean section among women delivering macrosomic babies was 47.6% (199, while 52.4% (219 delivered vaginally. Conclusion: Despite extensive efforts to reduce fetal and maternal complications associated with macrosomia, considerable fetal and maternal morbidity remain associated with this condition.

  8. Preparative scale production of functional mouse aquaporin 4 using different cell-free expression modes.

    Directory of Open Access Journals (Sweden)

    Lei Kai

    Full Text Available The continuous progress in the structural and functional characterization of aquaporins increasingly attracts attention to study their roles in certain mammalian diseases. Although several structures of aquaporins have already been solved by crystallization, the challenge of producing sufficient amounts of functional proteins still remains. CF (cell free expression has emerged in recent times as a promising alternative option in order to synthesize large quantities of membrane proteins, and the focus of this report was to evaluate the potential of this technique for the production of eukaryotic aquaporins. We have selected the mouse aquaporin 4 as a representative of mammalian aquaporins. The protein was synthesized in an E. coli extract based cell-free system with two different expression modes, and the efficiencies of two modes were compared. In both, the P-CF (cell-free membrane protein expression as precipitate mode generating initial aquaporin precipitates as well as in the D-CF (cell-free membrane protein expression in presence of detergent mode, generating directly detergent solubilized samples, we were able to obtain mg amounts of protein per ml of cell-free reaction. Purified aquaporin samples solubilized in different detergents were reconstituted into liposomes, and analyzed for the water channel activity. The calculated P(f value of proteoliposome samples isolated from the D-CF mode was 133 µm/s at 10°C, which was 5 times higher as that of the control. A reversible inhibitory effect of mercury chloride was observed, which is consistent with previous observations of in vitro reconstituted aquaporin 4. In this study, a fast and convenient protocol was established for functional expression of aquaporins, which could serve as basis for further applications such as water filtration.

  9. Cell-free protein expression based on extracts from CHO cells.

    Science.gov (United States)

    Brödel, Andreas K; Sonnabend, Andrei; Kubick, Stefan

    2014-01-01

    Protein expression systems are widely used in biotechnology and medicine for the efficient and economic production of therapeutic proteins. Today, cultivated Chinese hamster ovary (CHO) cells are the market dominating mammalian cell-line for the production of complex therapeutic proteins. Despite this outstanding potential of CHO cells, no high-yield cell-free system based on translationally active lysates from these cells has been reported so far. To date, CHO cell extracts have only been used as a foundational research tool for understanding mRNA translation (Lodish et al., 1974; McDowell et al., 1972). In the present study, we address this fact by establishing a novel cell-free protein expression system based on extracts from cultured CHO cells. Lysate preparation, adaptation of in vitro reaction conditions and the construction of particular expression vectors are considered for high-yield protein production. A specific in vitro expression vector, which includes an internal ribosome entry site (IRES) from the intergenic region (IGR) of the Cricket paralysis virus (CrPV), has been constructed in order to obtain optimal performance. The IGR IRES is supposed to bind directly to the eukaryotic 40S ribosomal subunit thereby bypassing the process of translation initiation, which is often a major bottleneck in cell-free systems. The combination of expression vector and optimized CHO cell extracts enables the production of approximately 50 µg/mL active firefly luciferase within 4 h. The batch-type cell-free coupled transcription-translation system has the potential to perform post-translational modifications, as shown by the glycosylation of erythropoietin. Accordingly, the system contains translocationally active endogenous microsomes, enabling the co-translational incorporation of membrane proteins into biological membranes. Hence, the presented in vitro translation system is a powerful tool for the fast and convenient optimization of expression constructs, the

  10. Cell-free synthesis system suitable for disulfide-containing proteins

    Energy Technology Data Exchange (ETDEWEB)

    Matsuda, Takayoshi [NMR Pipeline Methodology Team, RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045 (Japan); Cell-Free Technology Application Laboratory, RIKEN Innovation Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045 (Japan); Watanabe, Satoru [NMR Pipeline Methodology Team, RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045 (Japan); Kigawa, Takanori, E-mail: kigawa@riken.jp [NMR Pipeline Methodology Team, RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045 (Japan); Cell-Free Technology Application Laboratory, RIKEN Innovation Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045 (Japan); Department of Computational Intelligence and Systems Science, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8502 (Japan)

    2013-02-08

    Highlights: ► Cell-free synthesis system suitable for disulfide-containing proteins is proposed. ► Disulfide bond formation was facilitated by the use of glutathione buffer. ► DsbC catalyzed the efficient shuffling of incorrectly formed disulfide bonds. ► Milligram quantities of functional {sup 15}N-labeled BPTI and lysozyme C were obtained. ► Synthesized proteins were both catalytically functional and properly folded. -- Abstract: Many important therapeutic targets are secreted proteins with multiple disulfide bonds, such as antibodies, cytokines, hormones, and proteases. The preparation of these proteins for structural and functional analyses using cell-based expression systems still suffers from several issues, such as inefficiency, low yield, and difficulty in stable-isotope labeling. The cell-free (or in vitro) protein synthesis system has become a useful protein production method. The openness of the cell-free system allows direct control of the reaction environment to promote protein folding, making it well suited for the synthesis of disulfide-containing proteins. In this study, we developed the Escherichia coli (E. coli) cell lysate-based cell-free synthesis system for disulfide-containing proteins, which can produce sufficient amounts of functional proteins for NMR analyses. Disulfide bond formation was facilitated by the use of glutathione buffer. In addition, disulfide isomerase, DsbC, catalyzed the efficient shuffling of incorrectly formed disulfide bonds during the protein synthesis reaction. We successfully synthesized milligram quantities of functional {sup 15}N-labeled higher eukaryotic proteins, bovine pancreatic trypsin inhibitor (BPTI) and human lysozyme C (LYZ). The NMR spectra and functional analyses indicated that the synthesized proteins are both catalytically functional and properly folded. Thus, the cell-free system is useful for the synthesis of disulfide-containing proteins for structural and functional analyses.

  11. MR evaluation of fetal demise

    International Nuclear Information System (INIS)

    Fetal demise is an uncommon event encountered at MR imaging. When it occurs, recognition by the interpreting radiologist is important to initiate appropriate patient management. To identify MR findings of fetal demise. Following IRB approval, a retrospective search of the radiology fetal MR database was conducted searching the words ''fetal demise'' and ''fetal death.'' Fetuses with obvious maceration or no sonographic confirmation of death were excluded. Eleven cases formed the study group. These were matched randomly to live fetuses of similar gestational age. Images were reviewed independently by three pediatric radiologists. The deceased fetus demonstrates decreased MR soft-tissue contrast and definition of tissue planes, including loss of gray-white matter differentiation in the brain. The signal within the cardiac chambers, when visible, is bright on HASTE sequences from the stagnant blood; the heart is small. Pleural effusions and decreased lung volumes may be seen. Interestingly, the fetal orbits lose their anatomical round shape and become smaller and more elliptical; a dark, irregular rim resembling a mask may be seen. Although fetal demise is uncommonly encountered at MR imaging, radiologists should be aware of such imaging findings so prompt management can be instituted. (orig.)

  12. MR evaluation of fetal demise

    Energy Technology Data Exchange (ETDEWEB)

    Victoria, Teresa; Chauvin, Nancy Anne; Johnson, Ann M.; Kramer, Sandra Sue; Epelman, Monica [The Children' s Hospital of Philadelphia, Department of Radiology, Philadelphia, PA (United States); Capilla, Elena [Hospital Universitario Clinico San Carlos de Madrid, Madrid (Spain)

    2011-07-15

    Fetal demise is an uncommon event encountered at MR imaging. When it occurs, recognition by the interpreting radiologist is important to initiate appropriate patient management. To identify MR findings of fetal demise. Following IRB approval, a retrospective search of the radiology fetal MR database was conducted searching the words ''fetal demise'' and ''fetal death.'' Fetuses with obvious maceration or no sonographic confirmation of death were excluded. Eleven cases formed the study group. These were matched randomly to live fetuses of similar gestational age. Images were reviewed independently by three pediatric radiologists. The deceased fetus demonstrates decreased MR soft-tissue contrast and definition of tissue planes, including loss of gray-white matter differentiation in the brain. The signal within the cardiac chambers, when visible, is bright on HASTE sequences from the stagnant blood; the heart is small. Pleural effusions and decreased lung volumes may be seen. Interestingly, the fetal orbits lose their anatomical round shape and become smaller and more elliptical; a dark, irregular rim resembling a mask may be seen. Although fetal demise is uncommonly encountered at MR imaging, radiologists should be aware of such imaging findings so prompt management can be instituted. (orig.)

  13. Fetal valproate syndrome

    Directory of Open Access Journals (Sweden)

    Parmarth G Chandane

    2014-01-01

    Full Text Available Antenatal use of anticonvulsant valproic acid can result in a well-recognized cluster of facial dysmorphism, congenital anomalies and neurodevelopmental retardation. In this report, we describe a case with typical features of fetal valproate syndrome (FVS. A 26-year-old female with epilepsy controlled on sodium valproate 800 mg/day since 3 years, gave birth to a male child with characteristic features of FVS. She also had 3 spontaneous first-trimester abortions during those 3 years. Sodium valproate, a widely used anticonvulsant and mood regulator, is a well-recognized teratogen that can result in facial dysmorphism, craniosynostosis, neural tube defects, and neurodevelopmental retardation. Therefore, we strongly recommend avoidance of valproic acid and supplementation of folic acid during pregnancy.

  14. 21 CFR 884.2900 - Fetal stethoscope.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Fetal stethoscope. 884.2900 Section 884.2900 Food... Fetal stethoscope. (a) Identification. A fetal stethoscope is a device used for listening to fetal heart... conventional stethoscopes. (b) Classification. Class I (general controls). The device is exempt from...

  15. 高龄孕妇胎儿染色体非整倍体异常的早孕期检测%Detection of fetal chromosomal aneuploidy in pregnant women at advanced maternal age during the first trimester

    Institute of Scientific and Technical Information of China (English)

    汪淑娟; 高志英; 卢彦平; 李亚里; 姜淑芳; 汪龙霞; 张立文

    2014-01-01

    Objective To investigate the value of maternal plasma cell-free fetal DNA (cff-DNA) examination in detection of fetal chromosomal aneuploidy in pregnant women at advanced maternal ages during the first trimester of pregnancy. Methods A total of 136 pregnant women (11 to 13+6 gestational weeks) with advanced maternal ages were screened for fetal chromosomal aneuploidy with ultrasound and maternal plasma cff-DNA examination during March 1, 2011 to August 31, 2013, and the results were then confirmed by karyotype analysis and fluorescence in situ hybridization (FISH). Results Of the 136 women examined, cff-DNA screening detected chromosomal aneuploidy in 5 cases, including trisome-21 in 3 cases, trisome-18 in 1 case, and 45,X in 1 case as confirmed subsequently by karyotype analysis. Ultrasound screening reported a normal finding in one case of trisomy-21, thickening of the NT in the case of trisomy-18, and fetal anasarca in the case of 45,X. Karyotype analysis and follow-up of the women did not find chromosomal abnormality in the 131 negative cases screened by cff-DNA detection. Conclusion Screening of materal plasma cff-DNA allows accurate and early detection of fetal chromosomal aneuploidy in women of advanced maternal ages to avoid unnecessary invasive antenatal examinations.%目的:评价孕妇血浆胎儿游离DNA检测对高龄孕妇胎儿染色体非整倍体异常的早孕期检测价值。方法自2011年3月1日~2013年8月31日,采用早孕期超声筛查和孕妇血浆胎儿游离DNA检测技术,对136例孕11-13+6周的高龄孕妇进行检测,并通过染色体核型分析、荧光染色体原位杂交进行结果验证。结果cff-DNA检测,检出3例21-三体,1例18-三体(同时存在NT增厚),1例45,X(超声提示胎儿全身水肿),与荧光染色体杂交的结果一致,分别为47,XN,+21、47,XN,+18、45,X;筛查阴性者,未发现染色体异常。结论在早孕期开展cff-DNA检测,能更早期、

  16. MR imaging of fetal abnormalities

    International Nuclear Information System (INIS)

    We retrospectively reviewed 20 cases of fetal non-sedated MRI, in comparison with the findings at birth or at autopsy. In normal fetuses and cases of hydramnios, the MR Images poor with lots of motion artifacts. The MRI motion artifacts of anomalous fetus were less prominent than those of normal fetuses. In pregnancies with severe fetal anomalies and/or oligo-hydramnios, MR Images were more clear, and had less artifacts. MRI was more useful for prenatal evaluation of pregnancies with severe fetal anomalies. (author)

  17. MRI of the fetal abdomen

    International Nuclear Information System (INIS)

    Magnetic resonance imaging (MRI) is an important diagnostic component for central nervous system and thoracic diseases during fetal development. Although ultrasound remains the method of choice for observing the fetus during pregnancy, fetal MRI is being increasingly used as an additional technique for the accurate diagnosis of abdominal diseases. Recent publications confirm the value of MRI in the diagnosis of fetal gastrointestinal tract and urogenital system diseases. The following report provides an overview of MRI-examination techniques for the most frequent diseases of the abdomen. (orig.)

  18. Fetal homologue of infant crying

    OpenAIRE

    Gingras, J.; Mitchell, E; Grattan, K

    2005-01-01

    Four behavioural states are recognised in the human fetus and are comparable to those of the neonate: 1F (quiet sleep), 2F (active state), 3F (quiet awake), and 4F (active awake). State 5, or crying, is not considered to have a fetal correlate. In a study assessing the effects of exposure to tobacco and cocaine during pregnancy on fetal response and habituation to vibroacoustic stimulation, what appears to be the fetal homologue of crying was observed. These behaviours were seen on ultrasound...

  19. Maternal obesity affects fetal neurodevelopmental and metabolic gene expression: a pilot study.

    Directory of Open Access Journals (Sweden)

    Andrea G Edlow

    Full Text Available OBJECTIVE: One in three pregnant women in the United States is obese. Their offspring are at increased risk for neurodevelopmental and metabolic morbidity. Underlying molecular mechanisms are poorly understood. We performed a global gene expression analysis of mid-trimester amniotic fluid cell-free fetal RNA in obese versus lean pregnant women. METHODS: This prospective pilot study included eight obese (BMI≥30 and eight lean (BMI<25 women undergoing clinically indicated mid-trimester genetic amniocentesis. Subjects were matched for gestational age and fetal sex. Fetuses with abnormal karyotype or structural anomalies were excluded. Cell-free fetal RNA was extracted from amniotic fluid and hybridized to whole genome expression arrays. Genes significantly differentially regulated in 8/8 obese-lean pairs were identified using paired t-tests with the Benjamini-Hochberg correction (false discovery rate of <0.05. Biological interpretation was performed with Ingenuity Pathway Analysis and the BioGPS gene expression atlas. RESULTS: In fetuses of obese pregnant women, 205 genes were significantly differentially regulated. Apolipoprotein D, a gene highly expressed in the central nervous system and integral to lipid regulation, was the most up-regulated gene (9-fold. Apoptotic cell death was significantly down-regulated, particularly within nervous system pathways involving the cerebral cortex. Activation of the transcriptional regulators estrogen receptor, FOS, and STAT3 was predicted in fetuses of obese women, suggesting a pro-estrogenic, pro-inflammatory milieu. CONCLUSION: Maternal obesity affects fetal neurodevelopmental and metabolic gene expression as early as the second trimester. These findings may have implications for postnatal neurodevelopmental and metabolic abnormalities described in the offspring of obese women.

  20. Clinical relevance of circulating cell-free microRNAs in ovarian cancer.

    Science.gov (United States)

    Nakamura, Koji; Sawada, Kenjiro; Yoshimura, Akihiko; Kinose, Yasuto; Nakatsuka, Erika; Kimura, Tadashi

    2016-01-01

    Ovarian cancer is the leading cause of death among gynecologic malignancies. Since ovarian cancer develops asymptomatically, it is often diagnosed at an advanced and incurable stage. Despite many years of research, there is still a lack of reliable diagnostic markers and methods for early detection and screening. Recently, it was discovered that cell-free microRNAs (miRNAs) circulate in the body fluids of healthy and diseased patients, suggesting that they may serve as a novel diagnostic marker. This review summarizes the current knowledge regarding the potential clinical relevance of circulating cell-free miRNA for ovarian cancer diagnosis, prognosis, and therapeutics. Despite the high levels of ribonucleases in many types of body fluids, most of the circulating miRNAs are packaged in microvesicles, exosomes, or apoptotic bodies, are binding to RNA-binding protein such as argonaute 2 or lipoprotein complexes, and are thus highly stable. Cell-free miRNA signatures are known to be parallel to those from the originating tumor cells, indicating that circulating miRNA profiles accurately reflect the tumor profiles. Since it is well established that the dysregulation of miRNAs is involved in the tumorigenesis of ovarian cancer, cell-free miRNAs circulating in body fluids such as serum, plasma, whole blood, and urine may reflect not only the existence of ovarian cancer but also tumor histology, stage, and prognoses of the patients. Several groups have successfully demonstrated that serum or plasma miRNAs are able to discriminate patients with ovarian cancer patients from healthy controls, suggesting that the addition of these miRNAs to current testing regimens may improve diagnosis accuracies for ovarian cancer. Furthermore, recent studies have revealed that changes in levels of cell-free circulating miRNAs are associated with the condition of cancer patients. Discrepancies between the results across studies due to the lack of an established endogenous miRNA control to