Cell-free Fetal DNA In Plasma Research Articles

The current applications of cell-free fetal DNA in prenatal diagnosis of single-gene diseases: A review.

Prenatal diagnosis of hereditary diseases has substantially altered the way medical geneticists are helping families affected by genetic disorders. However, the risk of miscarriage and fear of invasive diagnostic procedures may discourage many couples from seeking prenatal diagnosis. With the discovery of maternal plasma cell-free fetal DNA, prenatal diagnosis has entered a new era of progress. Cell-free DNA is released during normal physiological functions as well as through the cell death programs of apoptosis and necrosis. It can be found in the plasma and other body fluids. Although this method has the advantage of being noninvasive, it is still rather expensive and requires advanced hardware and comprehensive data analysis. Promising implications of noninvasive prenatal diagnosis methods for the diagnosis of common trisomy disorders have paved the way for the development of more complicated assays of single-gene disorders. Relative mutation dosage and relative haplotype dosage are the most widely implemented assays for noninvasive prenatal diagnosis of single-gene disorders. However, each assay has its own advantages and disadvantages. Relative mutation dosage is based on the droplet digital polymerase chain reaction (PCR) technique which includes quantification features of real-time PCR assays. Relative haplotype dosage is based on next-generation sequencing that includes analysis of the maternal and paternal genome followed by sequencing of maternal plasma cell-free DNA. Co-amplification at a lower denaturation temperature PCR is another approach that is based on forming heteroduplexes between alleles to selectively amplify paternal mutations. In this review, we have described the most common noninvasive prenatal diagnosis approaches and compared their applications in genetic disorder diagnosis with different inheritance patterns.

Current controversies in prenatal diagnosis 3: is there still a value in a nuchal translucency screening ultrasound in conjunction with maternal plasma non-invasive cell-free DNA testing?

What's already known about this topic? Nuchal translucency is an ultrasound measurement of the fetal physiological lymphatic development as it can identify both normal and abnormal lymphatic collections. Abnormal or enlarged nuchal translucency measurements are nonspecific and heterogeneous in origin. The source of maternal plasma cell free fetal DNA is placental apoptosis. The embryonic and cellular development of the placenta can have true chromosomal mosaicism (involves fetus and placenta) orconfined placental mosaicism (CPM) (placenta only). CPM is present in 1–2% of placentas and can cause false positive results for diagnostic chorionic villus sampling and maternalplasma cell free DNA prenatal aneuploidy screening resultswhile false negative placental results are much less common at 1 per 3000. What does this study add? This report summarizes an oral debate presented at the 19th International Conference on Prenatal Diagnosis and Therapy inWashington, DC, USA, on 14 July 2015. This study compares the fetal aneuploidy screening and detection process with the ultrasound nuchal translucency aneuploidy-geneticpathology screening test/measurement and the more recent maternal plasma cell free DNA aneuploidy only screening test when used in conjunction or separately. Informed consent and patient understanding of the aneuploidy screening options and techniques is an important counselling principle for the variety of aneuploidy and genetic screening protocols.

Fetal RHD Genotyping from Circulating Cell-Free Fetal DNA in Plasma of Rh Negative Pregnant Women in Iran.

The prenatal determination of the fetal Rh genotype could lead to a substantial reduction in the use of anti-D immunoglobulin and prevention of unnecessary exposure of pregnant women carrying RhD negative fetus. The aim of this study was fetal RHD genotyping through the analysis of cffDNA in plasma samples of RhD negative pregnant women by real-time PCR technique. In this experiment, 30 plasma samples were collected from RhD negative pregnant women. DNA were extracted and real-time PCR reactions were done by specific primers for RHD, SRY and beta-globin (GLO) genes. The Rh phenotypes of mothers and their babies were determined by agglutination method and specific anti-serums. From the 30 maternal plasma samples considered for SRY genotyping, 16 samples revealed the presence of the SRY gene. Regarding the fetal RHD genotyping, 26 samples were positive for RhD and 4 samples were negative. In all cases, the predicted RhD and SRY genotypes were in concordance with the serologically determined phenotypes. The sensitivity, specificity and precision of the fetal RHD and SRY genotyping test were calculated 100% (p value <0.0005; K=100%). The present study confirms the precision of fetal RHD and SRY genotyping in maternal plasma by real-time PCR technique. This method helps RhD negative pregnant women about the appropriate use of anti-D immunoglobulin and also on the management and prevention of HDFN. However, superior and confirmatory studies are recommended before fetal RHD genotyping by real-time PCR is introduced as a non-invasive prenatal screening test.

Assessment of plasma cell-free fetal DNA using hypermethylated RASSF1A in maternal plasma in cases of spontaneous preterm labor

Objective The aim of this study was to determine the relation between the levels of cell-free fetal DNA (cffDNA) in maternal plasma and the occurrence of preterm labor. Materials and methods This was a prospective case–control study including 60 pregnant women between 28 and 32 weeks’ gestation, divided into a study group that included 30 patients with threatened or established preterm labor, and another 30 pregnant women with no symptoms or signs of preterm labor, representing the control group. cffDNA levels were quantified by real-time PCR using the RASSF1A gene before and after treatment by methylation-sensitive restriction enzyme, respectively, irrespective to fetal sex. Results The mean cffDNA levels in the study and control groups were 229.12±71.95 and 39.81±5.95 copies/ml, respectively. The cffDNA level was about six folds increased in group A (study group), with a statistical significance (P<0.05). Conclusion High levels of cffDNA between 28 and 32 weeks are associated with an increased risk of spontaneous preterm delivery.

Plasma circulating cell-free DNA and uteroplacental blood flow in pre-eclamptic patients

Objective The aim of the present study is to evaluate the plasma total cell-free DNA and cell-free fetal DNA level in maternal venous samples obtained from pre-eclamptic pregnant women during the third trimester (gestational age>28 weeks) and whether it shows a correlation with uteroplacental blood flow. Materials and methods This was a case–control study in which plasma total cell-free DNA, cell-free fetal DNA levels, and uteroplacental blood flow including umbilical artery resistance index, uterine artery resistance index and pulsitility index, and middle cerebral artery/umbilical artery pulsitility index were measured in 60 women diagnosed with pre-eclampsia, representing the study group, and another 60 normotensives were taken as controls. Both total cell-free DNA and cell-free fetal DNA were quantified by real-time PCR using the RASSF1A gene before and after treatment by methylation-sensitive restriction enzyme, respectively, irrespective of the sex of the fetus. Results Our results showed that there was a positive correlation between plasma cell-free fetal DNA, total cell-free DNA, and uteroplacental blood flow in pre-eclamptic women (P<0.05). To detect pre-eclampsia, the best cut-off value for plasma cell-free fetal DNA level was 80 copies/ml and that for plasma total cell-free DNA level was 137 copies/ml. Conclusion Our results found that there was a positive correlation between plasma cell-free fetal DNA level and the severity of pre-eclampsia. In addition, we found a correlation between plasma cell-free fetal, total cell-free DNA level, and uteroplacental blood flow in pre-eclamptic women during the third trimester.

Early non-invasive detection of fetal Y chromosome sequences in maternal plasma using multiplex PCR

Clinical indications for fetal sex determination include risk of X-linked disorders, a family history of conditions associated with ambiguous development of the external genitalia, and some fetal ultrasound findings. It is usually performed in the first trimester from fetal material obtained through CVS and is associated with an approximately 1% risk of miscarriage. Ultrasound fetal sex determination is often performed after 11 weeks of gestation. This study aims to validate a reliable method for non-invasive prenatal diagnosis of fetal gender using maternal plasma cell-free fetal DNA (cffDNA) for fetal sex assessment in the first trimester of pregnancy and test its clinical utility in the diagnosis of potentially affected pregnancies in carriers of X-linked disorders. In the validation study, blood samples from 100 pregnant women at 6-11 weeks of gestation were analysed. In the clinical study, 17 pregnancies at risk of having an affected fetus were tested. 7 ml of maternal blood in EDTA were obtained and cffDNA was extracted using a commercially available kit. DNA was enzymatically digested using a methylation sensitive endonuclease (AciI) to remove maternal unmethylated sequences of the RASSF1A gene. A multiplex PCR was performed for the simultaneous amplification of target sequences of SRY and DYS14 from chromosome Y, along with RASSF1A and ACTB sequences. Amplification of these loci indicates fetal gender, confirms the presence of cffDNA and allows assessment of digestion efficiency. After establishing the appropriate experimental conditions, validation studies were successful in all 100 cases tested with no false negative or false positive results. Y chromosome-specific sequences were detected in 68 samples, and 32 cases were diagnosed as female based on the amplification of RASFF1A sequences only, in the absence of ACTB. In the clinical studies, fetal sex was correctly diagnosed in 16 pregnancies, and one case was reported as inconclusive. Fetal sex assessment by detecting Y chromosome sequences in maternal blood can be routinely used from the 6th week of gestation. Reliable fetal sex determination from maternal blood in the 1st trimester of gestation can avoid conventional invasive methods of prenatal diagnosis.

Non-invasive prenatal diagnosis of down syndrome: a dream come true

Our group discovered the presence of cell-free fetal DNA in plasma of pregnant women. This finding offered opportunities for the development of non-invasive approaches for prenatal diagnosis. However, fetal DNA co-exists with a large background of maternal DNA and amounts to just some 10% of the total DNA in maternal plasma. Despite the technical challenges, we developed an approach based on massively parallel sequencing for the non-invasive detection of fetal Down syndrome by detecting the small increments in chromosome 21 DNA sequences in maternal plasma. We showed in a study of 750 pregnancies that Down syndrome can be accurately identified by direct analysis of maternal plasma DNA. The method could be further adapted for the prenatal detection of trisomy 18 and trisomy 13. A number of groups have since confirmed our findings. Non-invasive prenatal detection of fetal Down syndrome has since been implemented as a routine service at a number of centres around the world. <h3>Acknowledgement</h3> Supported by the University Grants Committee of the Government of the Hong Kong Special Administrative Region, China, under the Areas of Excellence Scheme (AoE/M-04/ 06), the General Research Fund Scheme of the Hong Kong Research Grants Council (CUHK463109), and an Innovation and Technology Fund (ITS/054/09).

Non-invasive prenatal diagnosis of down syndrome: a dream come true

Our group discovered the presence of cell-free fetal DNA in plasma of pregnant women. This finding offered opportunities for the development of non-invasive approaches for prenatal diagnosis. However, fetal DNA co-exists with a large background of maternal DNA and amounts to just some 10% of the total DNA in maternal plasma. Despite the technical challenges, we developed an approach based on massively parallel sequencing for the non-invasive detection of fetal Down syndrome by detecting the small increments in chromosome 21 DNA sequences in maternal plasma. We showed in a study of 750 pregnancies that Down syndrome can be accurately identified by direct analysis of maternal plasma DNA. The method could be further adapted for the prenatal detection of trisomy 18 and trisomy 13. A number of groups have since confirmed our findings. Non-invasive prenatal detection of fetal Down syndrome has since been implemented as a routine service at a number of centres around the world. <h3>Acknowledgement</h3> Supported by the University Grants Committee of the Government of the Hong Kong Special Administrative Region, China, under the Areas of Excellence Scheme (AoE/M-04/ 06), the General Research Fund Scheme of the Hong Kong Research Grants Council (CUHK463109), and an Innovation and Technology Fund (ITS/054/09).

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

Aneuploidy Detection in Mixed DNA Samples by Methylation-Sensitive Amplification and Microarray Analysis

Cell-free fetal nucleic acid, believed to be derived from the placenta/trophoblast, is present in the plasma of pregnant women; however, its use for predictive genetic testing has been severely limited because the circulating fetal DNA is present in a small quantity and mixed with a much larger quantity of maternal DNA. Methods for detecting fetal aneuploidy from the cell-free fetal DNA in plasma are highly sought after, but proposed methods must take into account the small quantity and highly contaminated nature of the available fetal DNA. We developed a method for methylation-sensitive amplification of DNA suitable for use with small (approximately 1 ng) samples. We used this method in conjunction with 2-color microarray analysis with a custom-made array to investigate whether relative amplification, and hence relative methylation, could be evaluated for a large number of genomic loci. Microarray assessment of genomic methylation accurately predicted the degree of methylation measured with bisulfite-conversion PCR and confirmed that DNA from first-trimester trophoblast was generally hypomethylated compared with whole-blood DNA. With a series of 3 samples in which 1 ng of DNA from a trisomic first trimester placenta was mixed with 9 ng of chromosomally normal peripheral blood DNA, we observed that the microarray signal associated with the trisomic chromosome was significantly different from that of the other chromosomes (P < 0.001). This method has potential to be used for noninvasive detection of fetal aneuploidy.

Improving Enrichment of Circulating Fetal DNA for Genetic Testing: Size Fractionation Followed by Whole Gene Amplification

Objective: Among the pitfalls of using cell-free fetal DNA in plasma for prenatal diagnosis is quality of the recovered DNA fragments and concomitant presence of maternal DNA (>95%). Our objective is to provide alternative methods for achieving enrichment and high-quality fetal DNA from plasma. Methods: Cell-free DNA from 31 pregnant women and 18 controls (10 males and 8 females) were size separated using agarose gel electrophoresis. DNA fragments of 100–300, 500–700 and 1,500–2,000 bp were excised and extracted, followed by whole genome amplification (WGA) of recovered fragments. Levels of β-globin and DYS1 were measured. Results: Distribution of β-globin size fragments was similar among pregnant women and controls. Among control male cases, distribution of size fragments was the same for both β-globin and DYS1. Among maternal cases confirmed to be male, the smallest size fragment (100–300 bp) accounted for nearly 50% (39.76 ± 17.55%) of the recovered DYS1-DNA (fetal) and only 10% (10.40 ± 6.49%) of β-globin (total) DNA. After WGA of plasma fragments from pregnant women, DYS1 sequence amplification was best observed when using the 100–300 bp fragments as template. Conclusions: Combination of electrophoresis for size separation and WGA led to enriched fetal DNA from plasma. This novel combination of strategies is more likely to permit universal clinical applications of cell-free fetal DNA.

First-trimester maternal plasma cell-free fetal DNA and preeclampsia

The purpose of this study was to determine whether, in pregnancies that experience preeclampsia, plasma cell-free fetal DNA (cffDNA) at 11-13 weeks of gestation is increased and whether this increase is related to the uterine artery pulsatility index (PI). Plasma cffDNA and uterine artery PI were measured in 44 cases with preeclampsia, which included 11 cases that required delivery at <34 weeks of gestation and 176 normal control subjects. All fetuses were male, and cffDNA was assessed by amplification of the DYS14 gene. The association between cffDNA and uterine artery PI was assessed by regression analysis. Median cffDNA was higher in early preeclampsia (median, 95.5 genome equivalents/mL; interquartile range, 72.7-140.9 genome equivalents/mL), but not late preeclampsia (median, 50.8 genome equivalents/mL; interquartile range, 25.0-103.8 genome equivalents/mL), than control subjects (median, 51.5 genome equivalents/mL; interquartile range, 31.1-84.9 genome equivalents/mL). There was a significant association between cffDNA and uterine artery PI (P = .038) but not in the control subjects (P = .174). The increase in plasma cffDNA in pregnancies that experience preeclampsia is associated with the degree of impairment in placental perfusion.

Presence of Cell-Free Fetal DNA in Plasma of Women with Ectopic Pregnancies,

The quantity of cell-free fetal DNA in the plasma of pregnant women changes during pregnancy and seems to be different in normal and pathologic pregnancies. We investigated the possible diagnostic applications of the detection and measurement of cell-free fetal DNA by comparing quantities found in women with ectopic (EP) or intrauterine (IUP) pregnancies. We collected blood samples from 58 women who had positive pregnancy tests and specific complaints and sonographic findings suggestive of EP and from 45 women with confirmed IUP. We performed quantitative real-time PCR analysis of the sex-determining region Y (SRY) gene to detect and measure the amount of cell-free fetal DNA. The diagnosis of EP was confirmed by histologic examination. SRY was detected in 15 EP and 14 IUP cases. The mean (SD) amount of cell-free fetal DNA was significantly higher (P<0.005) in women with EP [565 (136) genome-equivalents (GE)/mL] than in women with IUP [72 (19) GE/mL] at the same gestational age. Our results confirm that cell-free fetal DNA is present in plasma of women with EP. The finding of higher amounts of cell-free fetal DNA in EP cases than in IUP cases suggests that this method might be useful for early diagnosis of EP.

Placental volume, as measured by 3-dimensional sonography and levels of maternal plasma cell-free fetal DNA

Measurement of cell-free fetal (cff) DNA in maternal plasma may have clinical application in prenatal screening for fetal Down syndrome and preeclampsia. Little is known regarding the tissue of origin of these fetal-derived sequences. We tested the hypothesis that if the placenta is the major contributor of cff DNA, then an increased placental volume should be associated with higher maternal plasma cff DNA levels. We enrolled 143 pregnant women who underwent first-trimester placental volume measurement using 3-dimensional ultrasonography. Cff DNA in maternal plasma on the day of the scan was quantified by real-time polymerase chain reaction (PCR) amplification of a Y-chromosome sequence. The association between measured placental volume and maternal plasma cff DNA levels was analyzed along with relevant clinical variables. The median (25th, 75th percentiles) maternal plasma cff DNA level was 16.9 genome equivalents (GE)/mL (10.8, 28.7). Raw values were adjusted for gestational age and maternal body mass index. The median (25th, 75th percentiles) placental volume was 53.2 mL (43.0, 64.7), and median placental quotient (ratio of placental volume to fetal crown-rump length) was 1 mm2 (0.8, 1.1). Based on multivariate linear regression analyses, neither of the above placental measurements showed a significant association with maternal plasma cff DNA levels (P = .43 and .43, respectively). A modest association was found between plasma cff DNA levels and gravidity (P = .03). Our data did not show a significant association between either the placental volume or placental quotient, and maternal plasma cff DNA levels. We speculate that it is the extent of placental apoptosis that primarily affects the amount of cff DNA released into the maternal circulation.

Analysis of Cell-free Fetal DNA in Plasma and Serum of Pregnant Women

Sixty blood samples from pregnant women during gestational weeks 9-28 were investigated. Cell-free fetal DNA was extracted from maternal plasma or serum to be detected by nested PCR for determination of fetal gender. The SRY gene as a marker for fetal Y chromosome was detected in 34/36 women carrying a male fetus. In 3/24 women carrying female fetuses, the SRY sequence was also detected. Overall, fetal sex was correctly predicted in 91.7% of the cases. Therefore, the new, non-invasive method of prenatal diagnosis of fetal gender for women at risk of producing children with X-linked disorders is reliable, secure, and can substantially reduce invasive prenatal tests.

Fragmentation of cell-free fetal DNA in plasma and urine of pregnant women

We assessed the effect of freezing on fragmentation of fetal DNA in maternal plasma and differences in DNA fragmentation between plasma and urine from pregnant women. 1. We prepared seven kinds of real-time PCR assays to amplify different-sized amplicons targeting the SRY gene. Fragmentation of fetal DNA in maternal plasma was compared between new (n=10) and 4-year-old samples (n=10). 2. To investigate differences in fragmentation of fetal DNA between plasma and urine from pregnant women, we amplified three different-sized amplicons and compared DNA fragmentation between plasma and urine (n=7). 1. Relative concentrations of fetal DNA compared to a 63-bp amplicon in new samples were 53.1, 42.0, 9.2 and 2.0% (median) for PCR amplicons of 107, 137, 193 and 313 bp, respectively. Concentrations in 4-year-old samples were 70.4, 40.9, 11.9 and 2.3%, respectively. 2. Although fetal DNA in urine was not detected for 107- and 137-bp amplicons of the SRY sequence, fetal DNA using a 63-bp amplicon was detectable in five of seven cases (71.4%). Cell-free fetal DNA in maternal plasma is stable under cryopreservation at -20 degrees C for at least 4 years. Approximately, 60% of fetal DNA in maternal plasma was fragmented to <100-bp long, and fetal DNA in urine was further fragmented. Maternal urine may be usable for detection of fetal DNA, although smaller target size is more important for PCR amplification of fetal DNA in urine than in the analysis of plasma from pregnant women.

Fetal DNA in maternal plasma as a screening variable for preeclampsia. A preliminary nonparametric analysis of detection rate in low-risk nonsymptomatic patients.

Our objective was to examine whether plasma fetal DNA can be used as a screening variable in those women who developed preeclampsia but without any clinical symptom at the time of blood draw. Fetal DNA was extracted from 1.5-mL plasma samples, and the DYS14 gene was analyzed by real-time quantitative polymerase chain reaction. Plasma collected and frozen from six women were each paired with five matched control samples of identical specimen type from gravid women carrying a presumed normal male fetus. Matched rank-sum analysis and nonparametric receiver operating characteristic (ROC) curves analysis of estimated multiples of median (MoM) were used for calculating detection rate (DR) and false-positive rate (FPR). The mean observed rank of 5.08 in the cases was significantly higher than the expected 3.18 (p-value = 0.013). Pregnancies that will develop a preeclampsia exhibit 2.39-fold higher levels of maternal plasma cell-free fetal DNA compared to matched controls. DR was 33 and 50% at an FPR of 5 and 10% respectively. The estimated DR allows to consider fetal DNA as a potential variable to predict preeclampsia in a low-risk population. Further studies will be addressed to calculate a parametric statistical algorithm and to estimate a proper posterior risk of the disease by means of fetal DNA alone or combined with other markers.

Increased Cell-free Fetal DNA in Plasma of Two Women with Invasive Placenta

Invasive placenta, often involving placenta accreta, placenta increta, and placenta percreta, is a life-threatening complication of pregnancy characterized by invasion of placental villi into the underlying myometrium. Invasive placenta is usually diagnosed as a result of abnormalities encountered during removal of the placenta at delivery. Because of profuse hemorrhaging in the early postpartum period, blood transfusion or emergency hysterectomy is often required in patients with invasive placenta. Invasive placenta is also associated with high maternal morbidity and a high risk of mortality. Therefore, prenatal prediction of this disease would be of great clinical benefit. Recently, Lo et al. (1) discovered the presence of fetal DNA in maternal plasma. They developed a real-time quantitative PCR assay with which to measure the concentration of fetal DNA within maternal plasma. In addition, they analyzed the Y-chromosome-specific SRY sequence to quantify the number of genome equivalents per milliliter of plasma in women carrying male fetuses (2). Although cell-free fetal DNA is reported to increase in maternal plasma in cases of preterm delivery (3), preeclampsia (4)(5), and pregnancy with a trisomy 21 fetus (6), it is unclear where this fetal DNA originates. In the …

THE LEVELS OF CIRCULATORY FETAL DNA IN MATERNAL PLASMA ARE ELEVATED PRIOR TO THE ONSET OF PREECLAMPSIA

Objective: Elevations in cell free fetal DNA has previously been determined in pregnancies affected by preeclampsia. A recent report has indicated that cell free fetal DNA concentrations are elevated early in pregnancy before disease onset. As we have recently performed a prospective study to examine fetal cell traffic in pregnancies at risk for developing preeclampsia, we now quantify cell free fetal DNA concentrations in these samples. Methods: Blood samples were collected in the second trimester of pregnancy from pregnancies at risk for preeclampsia. Cell free fetal DNA amounts were quantified by real-time PCR. These results were then correlated with subsequent pregnancy outcome. Results: Free fetal DNA levels were significantly higher (median of 422.9 vs. 128.5 copies/mL maternal plasma; p=0.005) in plasma samples from women who developed preeclampsia (n=10) when compared to those who had unremarkable pregnancies (n=40). Conclusions: Our data independently confirm the finding that maternal plasma cell free fetal DNA levels are elevated early in pregnancies, which later develop preeclampsia.