Detection Of Cell-free Fetal DNA Research Articles

A study on maternal and fetal cell free DNA (cffDNA) for predicting the adverse pregnancy outcomes

This gender-independent detection of cell-free fetal DNA in maternal plasma using RASSF1A/beta-actin has curtained off a new dimension regarding its utility to predict the adverse pregnancy outcomes. Recent efforts have been directed at developing sequences from cell-free fetal DNA (cffDNA) as markers for pregnancy outcomes. The utility of cffDNA using the methylation-dependent DSCR3 and RASSF1A markers along with total cell-free DNA (cf-DNA) in maternal serum by HYP2 marker are useful in predicting adverse pregnancy outcomes. Indigenously developed low-cost method of the gender-independent sequence markers from cffDNA was investigated and evaluated with the standardized commercial kits as predictive markers for adverse pregnancy outcomes. In the present study, we have tested whether the elevated amount of cffDNA in maternal plasma is associated with adverse pregnancy outcomes and development of new marker by the low-cost method to predict adverse pregnancy outcomes. 210 pregnant women within the age group of 20 – 30 years attending for routine antenatal checkups after 20 weeks with fulfilling the diagnostic criteria of adverse pregnancy outcomes were included in our study. Age-matched pregnant women without adverse pregnancy outcomes were included as controls (n=210). Identification of cell-free fetal DNA (cffDNA) in maternal plasma by using in-house methods (Guanidium isothiocyanate) was found comparable with commercial kit and its content (GE/ µl) in adverse pregnancy outcomes subjects were significantly higher than the normotensive subjects. Our results indicated that indigenously developed method for detection of gender-independent cffDNA can be applicable for screening test of adverse pregnancy outcome.

Label-free E-DNA biosensor based on PANi-RGO-G*NPs for detection of cell-free fetal DNA in maternal blood and fetal gender determination in early pregnancy

In this study, a DYS14 aptamer/polyaniline–reduced graphene oxide–gold nanoparticles/gold (Apt/PANi–RGO–G*NPs/Au) electrode was fabricated to detect the Y-chromosome DYS14 DNA sequence in cffDNA in the blood plasma of pregnant women and used on real and laboratory samples with high success rate. The electrochemical properties of the prepared E-DNA biosensor were characterized by CV, SWV, XRD, and EIS. The E-DNA biosensor morphological characteristics were investigated by TEM, SEM, and EDX. Phosphorothioate was used to link the aptamer to PANi–RGO–G*NPs modified gold electrode. This is due to control of the adsorption polarity and increase adsorption stability. Under optimized conditions, the linear range of the analytical technique with respect to the logarithm of the target sequence concentration was 1.0 × 10−16–1.0 × 10−8 M, the detection limit was 4.26 × 10−17 M, and the limit of quantitation was 1.422 × 10−16 M. The E-DNA biosensor displayed high selectivity and sensitivity, high efficiency, and acceptable repeatability. For fetal sex detection, 12 pregnant women from the 5th to the 15th week of gestation participated in the study. Results indicated the fabricated Apt/PANi–RGO–G*NPs/Au E-DNA biosensor to be appropriate for fetal sex determination in pregnant women between the 7th and 9th week of gestation. Notably, this method can be used as a model for the study of pathogens like bacteria and viruses.

The Role of Cell-Free Fetal DNA as a Preventative Attempt to Decrease the Severity of Genetic Disorders: A Review

The development of prenatal diagnosis has developed very rapidly over decades. Prenatal screening isapplied to a mother with high-risk pregnancy (threatening the life of the fetus), such as a too-old mother ora too-young mother and having a certain disease in her medical history. Up until now, the curative methodto treat genetic disorder is still developed and perfected. Therefore, a preventative action during pregnancyis important to do, such as Noninvasive Prenatal Testing (NIPT). NIPT method that is widely developedis the circulating cell-free fetal DNA (cffDNA) testing that can be useful for determining the fetal gender,the identification of specific single-gene disorders, blood type, paternity determination, and the potency ofregular use for testing chromosomal abnormalities. The detection of cell-free fetal DNA in the maternal bloodcirculation provides a new expectation for NIPT, especially preventing the severity of genetic disorders.

Detection of cell-free fetal DNA in maternal plasma using two types of compound markers.

With the discovery of circulating cell-free fetal DNA (cffDNA) in maternal plasma, noninvasive prenatal testing became possible. However, analysis of low-level cffDNA against high background maternal DNA remains complicated and challenging. To circumvent this limitation, selective amplification of cffDNA was used in this study. Two kinds of compound markers (namely DIP-STR and SNP-STR), both based on selective amplification, were used here for targeting fetal DNA. By designing two allele-specific forward primers for DIP-STR and SNP-STR, DNA fragments with different DIP/SNP alleles can be selectively amplified. When analyzing maternal plasma DNA, these markers can selectively target paternally inherited fetal alleles whose DIP/SNP allele was not shared with the mother. In this study, 21 families were studied with six DIP-STRs and 11 SNP-STRs. Fetal DNA was successfully detected across plasma samples for at least one marker. Detection rate varied between DIP-STR and SNP-STR markers, and DIP-STR outperforms SNP-STR. Fetal alleles obtained from maternal plasma were double confirmed by genotyping paternal genomic DNA and fetal genomic DNA from amniocentesis. This study demonstrated that selective amplification strategy can be used to target cffDNA in maternal plasma, which will be a promising method for noninvasive prenatal paternity testing.

Liquid biopsy for management of haemolytic disease of the fetus and newborn

Background: Haemolytic disease of the fetus and newborn arises from maternal allo-antibodies to red cell antigens, crossing the placenta. The most frequent antibodies after RhD, are Kell, Duffy and RhCE antigens. Liquid biopsy in pregnancy management is concerned with the detection of cell-free fetal DNA (cffDNA) circulating in the maternal plasma. The accuracy for fetal RHD genotyping is well established. Aim: To evaluate a maternal blood test to predict the fetal, non-RhD, blood group status. Methods: A clinical collaborative study assessed the accuracy for a suite of assays to predict fetal Kell, Duffy, Rhc/C and Rhe/E status for women with respective antibodies (n=90, gestation age 8–37 weeks). Droplet digital PCR technology, ddPCR, tested for single nucleotide variants (SNVs) associated with these blood group antigens. Infant cord blood serotypes were used as the primary outcome measure. Results: Fetal specific signals were detected as early as 8 weeks gestation. Genotyping matched all available infant cord outcomes 45/45. Accuracy is 100% (95% confidence interval 91.96–100%). Discussion: A clinical demand exists for liquid biopsy of maternal blood for fetal blood group genotyping. Droplet digital PCR is accurate and provides an evidence base to guide clinical care for at-risk pregnancies.

A Prospective study to evaluate the demographic variation of gender independent sequences in cell-free fetal DNA (cffDNA) concentration and to predict pregnancy outcomes by non-kit based economical method

This gender-independent detection of cell-free fetal DNA in maternal plasma using RASSF1A/β-actin has curtained off a new dimension regarding its utility to predict the adverse pregnancy outcomes. Recent efforts have been directed at developing sequences from cell-free fetal DNA (cffDNA) as markers for pregnancy outcomes. The utility of cffDNA using the methylation-dependent DSCR3 and RASSF1A markers along with total cell-free DNA (cf-DNA) in maternal serum by HYP2 marker are useful in predicting adverse pregnancy outcomes. Increased amount (>95th percentile) of cffDNA fraction in the second trimester is associated with preterm birth. Indigenously developed low-cost method of the gender-independent sequence markers from cffDNA was investigated and evaluated with the standardized commercial kits as predictive markers for adverse pregnancy outcomes. Our results indicated that indigenously developed method for detection of geneder-independent cffDNA can be applicable for screening test of adverse pregnancy outcome.

Quantitative detection of cell-free fetal DNA in peripheral blood of pregnant women during early pregnancy

Objective: This study aimed for the quantitative detection of cell-free fetal DNA (cffDNA) in peripheral plasma of pregnant women, which provides basic data for clinical non-invasive prenatal screening in early pregnancy. Materials and Methods: A total of 243 individuals with gestational age of 5-10+6 weeks were selected for this study, who required abortion. mDNA was extracted from villi, and Y-chromosome specific SRY gene was detected by nested PCR, which was regarded as standard for quantitative detection of accuracy. CffDNA was extracted from peripheral blood and SRY gene’s expression was detected by real-time fluorescent quantitative PCR. Results: Among 243 cases, nested PCR detected 163 individuals with SRY gene positive, and 80 were negative. Out of 163, in 140 cases SRY was detected in cffDNA, but 23 cases with gestational age 5-6+6 weeks were negative. However at 7th week of pregnancy, it was also detected in those 23 cases. The average concentration of cff DNA in 10th week of pregnancy was found significantly higher than in 7th, 8th, and 9th week of pregnancy. Conclusion: Thus, this study indicates the efficiency and reliability of cffDNA in peripheral blood of 7-10th week of gestational period for the detection of early pregnancy.

Clinical results after the implementation of cell-free fetal DNA detection in maternal plasma.

Detection of cell-free fetal DNA in maternal blood is a type of noninvasive prenatal diagnosis test (NIPT), which has already been known for some time but has not yet been introduced in most of public hospitals in Spain. How the implementation of cell-free fetal DNA (cffDNA) in a contingent protocol has influenced the aneuploidy screening in our hospital is described. Two cohorts of patients with positive combined screening were compared: the first one (years 2012-2013, 5747 patients) from a period of time in which the protocol valid until March 2016 - that included the use of invasive procedures - was applied; and the second one in which the current protocol - that included NIPT versus invasive procedures - was applied (first 7 months after protocol implementation, 898 patients). Comparison of both periods resulted in a 60.5% reduction of invasive procedures (P < 0,001) preserving the same chromosomopathy detection rate. The ratio of positive invasive procedures-indicated invasive procedures was improved by 15% in the first period to 50% in the second period (P = 0.01). NIPT introduction has caused a significant reduction of 60.5% of IP in high chromosomopathy risk patients after combined screening without modifying detection rate.

Cross-cultural perspectives on decision making regarding noninvasive prenatal testing: A comparative study of Lebanon and Quebec

ABSTRACTNoninvasive prenatal testing (NIPT), based on the detection of cell-free fetal DNA in maternal blood, has transformed the landscape of prenatal care by offering clinical benefits (noninvasive, high specificity and sensitivity, early detection of abnormalities) compared to existing prenatal screening tests. NIPT has expanded rapidly and is currently commercially available in most of the world. As NIPT spreads globally, culturally sensitive and ethically sound implementation will require policies that take into consideration the social and cultural context of prenatal testing decisions. In a Western context, the main ethical argument for providing access and public funding of prenatal tests is the promotion of reproductive autonomy (also referred to as “procreative liberty” and “reproductive freedom”), by enabling pregnant women and couples to access information about the fetus in order to choose a certain course of action for pregnancy management (continuation of pregnancy and preparation for birth or termination). So how is the framework of reproductive autonomy operationalized in non-Western cultural contexts? We used Quebec, Canada, and Beirut, Lebanon, for case studies to explore what ethical considerations related to reproductive autonomy should guide the implementation of the test in various cultural contexts. To answer this question, we conducted a qualitative study to (1) explore the perceptions, values, and preferences of pregnant women and their partners about NIPT and (2) examine how these values and perceptions influence reproductive autonomy and decision making in relation to NIPT in these two different cultural settings, Lebanon and Quebec. Our findings may guide health care professionals in providing counseling and in helping women and their partners make better informed prenatal testing decisions. Further, at a policy level, such understanding might inform the development of local guidelines and policies that are appropriate to each context.

Congenital Adrenal Hyperplasia: Unresolved Issues.

Congenital adrenal hyperplasia (CAH) describes a family of disorders that comes from enzymatic deficiencies in cortisol production, with 21-hydroxylase deficiency causing ∼90% of cases. Distinction is made between the severe classical form and milder nonclassical form of CAH. Molecular genetic analysis is used to confirm the hormonal diagnosis. A high rate of genotype-phenotype disconcordance has been found in 21-hydroxylase deficiency. The goal of treatment is to replace with synthetic glucocorticoids and mineralocorticoids and suppress adrenal androgen production. The treatment of patients affected with nonclassical CAH, particularly males, remains controversial. Variable synthetic glucocorticoids are used and new modes of glucocorticoid delivery are under investigation. To improve height, growth hormone and other adjuvant therapies are employed. Long-term outcomes of genital surgery using modern techniques in females affected with classical CAH continue to be investigated. Prenatal treatment with dexamethasone is available to avoid ambiguous genitalia in these females. Although studies have shown its safety to mother and fetus, prenatal treatment is still regarded as experimental. Currently, prenatal diagnosis of CAH can only be obtained through invasive methods. Recently, the detection of cell-free fetal DNA in maternal plasma has made it possible to make this diagnosis earlier and noninvasively.

Mrassf1a-Pap, a Novel Methylation-Based Assay for the Detection of Cell-Free Fetal DNA in Maternal Plasma

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.MethodsBisulfite sequencing was performed on maternal genomic (g)DNA 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.ResultsSerial 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%).ConclusionsOur 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 value as an addition to current techniques used in noninvasive prenatal diagnostics.

Value of detection of cell-free fetal DNA in maternal plasma in the prenatal diagnosis of chromosomal abnormalities

To investigate the value of detection of fetal cell-free fetal DNA (cff-DNA) in maternal plasma in the prenatal diagnosis of chromosomal abnormalities. The plasma from 3200 gravidas (singleton with 20.3 ± 3.8 gestational weeks) was collected from April 1(st) 2011 to May 30(th) 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. (1) The 3200 cases took cff-DNA detection, and 31 cases got positive results, including 27 cases of trisomy 21 and 4 cases of trisomy 18. Sixteen cases of trisomy 21 and 1 case of trisomy 18 were in the high-risk serological screening group. 7 cases of trisomy 21 and 2 cases of trisomy 18 were in the advanced age group. Four cases of trisomy 21 and 1 case of trisomy 18 were in the supplementary group. (2) And the 84% (26/31) cff-DNA detecting positive cases received amniocentesis. In the 27 trisomy 21 positive cases, 23 received amniocentesis and got karyotype of 47XN, +21, with the diagnostic accordance rate of 100%. In the 4 cases who didn't take karyotype analysis, fetal anomaly (ventricular septal defect, dextrocardia and choroid plexus cyst) was found in 1 case before 20 gestational weeks; intrauterine fetal demise happened in 1 case before getting the result; 2 other cases who already had healthy children took abortion in the local hospital without taking amniocentesis. In the 4 trisomy 18 positive cases, 3 took amniocentesis, 2 of which were trisomy 18 and took abortion, the other was chimera (46, XN/47, XN, +18) with only 2% cells of trisomy 18, with no malformation found after delivery. Hypoevolutism (3 weeks less than gestational week), general hydropsy and intrauterine fetal demise happened before the other case took amniocentesis. (3) Follow up of cff-DNA negative cases:until May 30(th) 2012, no Down's baby was found in the 1230 cases with cff-DNA test negative results. (1) The non-invasive fetal trisomy test (NIFTY) by next generation sequencing is a safe, accurate and high throughput method for the prenatal diagnosis of trisomy-21. (2) Use NIFTY as a further screening for pregnant women with high-risk serological screening results could lower invasive prenatal diagnosis rate. (3) Cases with positive NIFTY test results should receive amniocentesis and karyotype analysis to confirm the diagnosis before abortion.

Detection of cell-free fetal DNA in plasma of pregnant women using multiplex quantitative fluorescent-PCR

Detection of cell-free fetal DNA in plasma of pregnant women using multiplex quantitative fluorescent-PCR

Cell-Free DNA in Maternal Plasma: Has the Size-Distribution Puzzle Been Solved?

The detection of cell-free fetal DNA from maternal plasma and serum by Dennis Lo and colleagues at Oxford University, UK, opened an exciting route for the noninvasive determination of fetal genetic traits (1). It has proved very effective for identifying fetal genetic loci completely absent from the maternal genome, such as those on the Y chromosome or the RHD 3 (Rh blood group, D antigen) gene (2). Consequently, this approach is already being used in clinical settings for the determination of fetal sex in pregnancies at risk for X chromosome–linked disorders or fetal Rh D status in pregnancies at risk for hemolytic disease of the newborn. To date, several thousand analyses have been performed in several European centers. Commercial services are in the process of being launched in the US. The detection of fetal genetic loci less disparate from the maternal genome, such as point mutations or those involved in fetal aneuploidy, is more complex because of the preponderance of maternal cell-free DNA sequences (>90% of total cell-free DNA) (3). To overcome this problem, studies have undertaken to determine whether physical differences exist between maternal and fetal cell-free DNA fragments, specifically whether the fragments exhibit different size distributions. In the first of these …

Detection of Increased Amounts of Cell-Free Fetal DNA with Short PCR Amplicons

A digital PCR approach has recently been suggested to detect greater amounts of cell-free fetal DNA in maternal plasma than conventional real-time quantitative PCR (qPCR). Because the digital qPCR approach uses shorter PCR amplicons than the real-time qPCR assay, we investigated whether a real-time qPCR assay appropriately modified for such short amplicons would improve the detection of cell-free fetal DNA. We developed a novel universal-template (UT) real-time qPCR assay that was specific for the DYS14 sequence on Y chromosome and had a short amplicon size of 50 bp. We examined this "short" assay with 50 maternal plasma samples and compared the results with those for a conventional real-time qPCR assay of the same locus but with a longer amplicon (84 bp). Qualitatively, both assays detected male cell-free fetal DNA with the same specificity and detection capability. Quantitatively, however, the new UT real-time qPCR assay for shorter amplicons detected, on average, almost 1.6-fold more cell-free fetal DNA than the conventional real-time qPCR assay with longer amplicons. The use of short PCR amplicons improves the detection of cell-free fetal DNA. This feature may prove useful in attempts to detect cell-free fetal DNA under conditions in which the amount of template is low, such as in samples obtained early in pregnancy.

Early detection of cell-free fetal DNA in maternal plasma

Objectives We aimed to establish the earliest gestational age at which fetal DNA in maternal plasma could be detected and whether this was reliable at 12–13 weeks' gestation. Study design A prospective observational cohort study of 32 pregnancies either after IVF or before prenatal diagnosis by CVS. Maternal blood was taken and RT-PCR was carried out to detect the multi-copy Y chromosome associated DSY14 gene. The end point was gender as assessed at delivery or on karyotype. Results Y signal was obtained as early as 14 days post conception (4 weeks' gestation) and has a good prediction rate by 12 weeks' gestation. Conclusion Free fetal DNA allows very early prediction of fetal sex in some cases and could be useful for clinical use for X-linked conditions by the end of the first trimester.

Detection of cell-free fetal DNA in maternal urine

To evaluate the presence of cell-free fetal DNA signals in maternal urine as a potential source of material for non-invasive prenatal diagnosis. Patients referred to the regional fetal medicine unit who underwent prenatal diagnosis by chorionic villus sampling (CVS) were asked to give blood and urine immediately before the procedure. Maternal blood and urine were centrifuged at 10,000 g for 10 min. Plasma (1 mL) and urine (1 mL) supernatant were transferred to a clean tube and centrifuged again. The plasma (0.8 mL) and urine (0.8 mL) supernatant were removed without disturbing the cell pellet and stored at - 80 degrees C. Following DNA extraction, each sample was tested for the presence of Y chromosome associated DYS14 gene using real-time polymerase chain reaction (PCR). The total amount (maternal and fetal) of DNA in each sample was estimated using a quantitative real-time PCR assay. Twenty patients were enrolled in the study. CVS was performed at a median gestational age of 13 weeks (range 11 + 5 - 14 + 1). There were 12 male and 8 female fetuses, as confirmed by karyotype. Y chromosome DNA was not detected in any of the 20 samples of maternal urine, including 12 of the 20 samples in which Y chromosome DNA was detected in maternal plasma (all of whom were subsequently confirmed to be carrying a male fetus). There was considerable variation in the amount of total free DNA detected in maternal urine. Cell-free fetal DNA either was not present or did not amplify in maternal urine.

Detection of donor-specific DNA polymorphisms in the urine of renal transplant recipients.

Recently, a novel form of chimerism, termed urinary DNA chimerism, has been described in kidney transplant recipients in that cell-free DNA from the donor kidney was detected in the recipient’s urine (1). Quantitative analysis of this urinary donor-derived DNA has indicated that it may serve as a new marker to monitor kidney transplant engraftment because increased concentrations were present under conditions of graft rejection, which decreased to basal values after immunosuppressive treatment (2). A caveat of these studies was that they relied on sex-disparate donor–recipient conditions: because the PCR assays used were specific for the Y chromosome, cell-free DNA from the donor kidney could be detected only in the urine of female recipients who had received male kidneys (1)(2). We examined whether other kidney donor-derived DNA sequences could be detected in the urine of transplant recipients, using PCR assays specific for highly polymorphic short tandem repeat (STR) loci, also termed microsatellite markers. Previous examinations using such polymorphic genetic loci have shown that they can be used for differentiating female fetal cells from maternal ones (3)(4) or for the gender-independent detection of cell-free fetal DNA in maternal plasma (5)(6). For this purpose, we tested for the presence of donor-specific STR loci in the urine of cases in which the donor and recipient were either of the same sex or the donor was female and the recipient was male. For our study, which was approved by our respective ethics review boards, five cases involving living-donor (four related and one unrelated) transplants were enrolled. Blood samples …