Fetal DNA In Maternal Blood Research Articles

A Pilot Study of Noninvasive Prenatal Diagnosis of Alpha- and Beta-Thalassemia with Target Capture Sequencing of Cell-Free Fetal DNA in Maternal Blood.

Thalassemia is a dangerous hematolytic genetic disease. In south China, ∼24% Chinese carry alpha-thalassemia or beta-thalassemia gene mutations. Given the fact that the invasive sampling procedures can only be performed by professionals in experienced centers, it may increase the risk of miscarriage or infection. Thus, most people are worried about the invasive operation. As such, a noninvasive and accurate prenatal diagnosis is needed for appropriate genetic counseling for families with high risks. Here we sought to develop capture probes and their companion analysis methods for the noninvasive prenatal detection of deletional and nondeletional thalassemia. Two families diagnosed as carriers of either beta-thalassemia gene or Southeast Asian deletional alpha-thalassemia gene mutation were recruited. The maternal plasma and amniotic fluid were collected for prenatal diagnosis. Probes targeting exons of the genes of interest and the highly heterozygous SNPs within the 1Mb flanking region were designed. The target capture sequencing was performed with plasma DNA from the pregnant woman and genomic DNA from the couples and their children. Then the parental haplotype was constructed by the trios-based strategy. The fetal haplotype was deduced from the parental haplotype with a hidden Markov model-based algorithm. The fetal genotypes were successfully deduced in both families noninvasively. The noninvasively constructed haplotypes of both fetuses were identical to the invasive prenatal diagnosis results with an accuracy rate of 100% in the target region. Our study demonstrates that the effective noninvasive prenatal diagnosis of alpha-thalassemia and beta-thalassemia can be achieved with the targeted capture sequencing and the haplotype-assisted analysis method.

Sex determination using free fetal DNA in early pregnancy: With the approach to sex linked recessive disorders

Introduction: Prenatal diagnosis is testing for detection of diseases or conditions in a fetus or embryo before it is born. Most of prenatal diagnostic (PD) techniques are invasive and done in late stages of pregnancy. Using fetal DNA in maternal blood for fetal sex determination in early pregnancy might help in management of X-linked genetic diseases. This study aimed to investigate the accuracy of sex determination using fetal DNA in maternal blood at 8-12 weeks of gestation. Methods: In this cross-sectional study, 30 pregnant women at 8-12 weeks of gestation were enrolled. The sex-determining region Y (SRY) gene expression with the internal control (IC) glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was investigated with quantitative real-time polymerase chain reaction (PCR) using specific primers and probes. Results: Accuracy of sex determination with SRY gene expression in 8-12 weeks of pregnancy were 85%, 85%, 90% and 100% respectively. Conclusion: It seems that fetal sex determining using fetal DNA in maternal blood is a reliable method for early stage of pregnancy.

VP36 Cost-Effectiveness Of Non-Invasive Prenatal Testing For Down Syndrome

INTRODUCTION:The analysis of cell-free fetal DNA in maternal blood, also called Non-Invasive Prenatal Testing (NIPT), represents an emerging technology and a possible alternative/complement to current prenatal screening based on biochemical and sonographic markers for Down Syndrome (DS) detection.The aim of the study was to compare the application of NIPT with the prenatal diagnosis/screening procedures currently applied in the Basque Country.METHODS:An analytical decision model was developed to assess the costs and consequences, comparing current prenatal screening, NIPT as a contingency test in high-risk cases and NIPT as a first-line screening test. An economic analysis was conducted to determine which strategy was more cost-effective. Sensitivity analyses were performed (1).RESULTS:For a population of 97,074 pregnant women in gestational week 14 and a cut-off point of 1:270, NIPT as a contingent test was not cost-effective, detecting two cases less of DS and causing a lower number of miscarriages related to invasive-testing (4 versus 23) at a slightly lower cost (EUR8,111,351 versus EUR8,901,872).For risk cut-off points of 1:500 or 1:1000 for contingent NIPT, the number of DS cases detected increased, as did the cost. It could be cost-effective compared with current prenatal screening, (EUR61,763 or EUR256,123 per extra DS case detected, respectively).Using the NIPT as a primary test detected more DS cases (296 versus 271) and caused less miscarriages (5 versus 23), at a substantially higher cost (EUR41,395,645 versus EUR8,901,872). Cost-effectiveness analysis indicated that it was more expensive and more effective.Univariant sensitivity-analysis showed that when the price of the NIPT as primary test was EUR76, it was dominant compared with current prenatal screening. It was also cost-effective compared with the NIPT as a contingent test (EUR9,869 per extra DS case detected).CONCLUSIONS:The study shows that NIPT had higher detection rates for DS in different scenarios, but the cost constitutes a limiting factor for implementation in the Basque Health System.

Duo test and aneuploidy detection in women under 35 years of age with high-risk pregnancy at the Hospital General de México

Non-invasive procedures for prenatal diagnosis are the most frequently used methods in the first trimester of pregnancy in pregnant women under 35 years of age because they do not involve risk of pregnancy loss; however, they are not considered to be a definitive diagnosis method. During the first trimester the duo test (PAPP-A and free βhGC), cell-free fetal DNA in maternal blood and structural ultrasound scans are the principal tools used; the quadruple marker test (αFP, E3, β-hCG, inhibin A) is used in the second trimester. However, the definitive diagnosis is performed by cytogenetic analysis through amniocentesis. MethodsThirty women, under 35 years of age with high-risk pregnancy, were studied with duo test and structural ultrasound in the first trimester and amniocentesis in weeks 15–18. ResultsOnly five duo tests were positive: three showed risk of trisomy 18 and one of Turner syndrome, they all corroborated with the cytogenetic study; the fifth showed a risk of Down's syndrome, however it was a chromosomally normal product. Three patients with a negative duo test cytogenetically detected with karyotypes with structural abnormalities, which were: deletion of the short arm of chromosome 18 [46,XY, del(18)(p11)], Robertsonian translocation between chromosome 13 and 14 [45,XY, rob(13:14)] and a chromosome derived from X [46,X, der(X)]. The duo test is a very useful tool for the diagnosis of numerical chromosome abnormalities, but not for detecting structural chromosome aberrations. However, it is essential to perform amniocentesis to definitively rule out chromosomal aberrations in products of conception.

Noninvasive prenatal testing beyond genomic analysis: what the future holds.

The discovery of cell-free fetal DNA in maternal blood enabled the development of DNA-based noninvasive prenatal testing. Noninvasive prenatal testing for chromosomal aneuploidy detection was first applied for clinical use a few years ago, resulting in a paradigm shift in prenatal testing. Apart from the use of cell-free fetal nucleic acids for the detection of fetal genetic or chromosomal diseases, we predict that the analysis of cell-free placental RNA and DNA methylation signatures would allow the noninvasive monitoring of placental function. These developments would potentially allow the screening and identification of a range of pregnancy-associated diseases, providing a holistic approach to prenatal management. This article covers the advancement of techniques in measuring cell-free fetal RNA and fetal-specific methylation patterns in maternal blood. Recently, genome-wide fetal transcriptome and methylome can be obtained from maternal plasma, which allow the identification of novel biomarkers and the elucidation of the pathogenesis of maternal and fetal diseases. In fact, some studies demonstrated the feasibility of applying the RNA and DNA methylation analysis techniques for prenatal disease assessment. This study reviews the evidence that demonstrates the potential utilities of cell-free fetal transcriptomic and methylomic analysis for the future assessment of pregnancy-associated disorders.

Algorithm-Based Fetal Gender Determination Using X and Y Mini-Short Tandem Repeats at Early Gestational Ages

Background: Detection of fetal DNA in maternal blood has been examined by many research groups for a few years; thereby, scientists have a shorter way to take to approach prenatal diagnosis of abnormal pregnancies. The Y chromosome sequences have recently become the most common applicable indices for fetal sex determination. Objectives: We conducted an algorithmic X and Y mini-Short Tandem Repeats (STRs) genotyping method that could solve the problem of false negative (no detection of Y sequences) results of previous methods. Patients and Methods: Blood samples were obtained from 106 pregnant women and their spouses. Conventional PCR amplified 19 mini-Short Tandem Repeats (STRs) and three non-STR markers, which were subsequently genotyped by the means of Polyacrylamide gel electrophoresis (PAGE). Results: Sensitivity and specificity of the mini-STR genotyping method for fetal DNA detection were calculated (95.9% and 98%, respectively) with a confidence interval of 95%. In addition, sensitivity and informativeness were computed for each of the single mini-STR markers in our conventional PCR method. We also introduced the minimum number of mini-STRs needed to reach maximum validity for fetal gender determination in clinical settings. Conclusions: Algorithm-based mini-STR genotyping method significantly increases the reliability (sensitivity and specificity) of gender determination using free fetal DNA and could be applied in prenatal clinical testing.

Effective use of the TSPY gene-specific copy number in determining fetal DNA in the maternal blood of cynomolgus monkeys.

Since the available concentration of single-copy fetal genes in maternal blood DNA is sometimes lower than detection limits by PCR methods, the development of specific and quantitative PCR detection methods for fetal DNA in maternal blood is anticipated, which may broaden the methods that can be used to monitor pregnancy. We used the TaqMan qPCR amplification for DYS14 multi-copy sequence and the SRY gene in maternal blood plasma (cell-free DNA) and fractional precipitated blood cells (cellular DNA) from individual cynomolgus monkeys at 22 weeks of pregnancy. The availability of cell-free fetal DNA was higher in maternal blood plasma than that of cellular DNA from fractional precipitated blood cells. There was a significantly higher (P < 0.001) mean copy number of fetal male DYS14 from maternal plasma (4.4 × 10(4) copies/mL) than that of detected fetal cellular DNA from fractional blood cell pellets. The sensitivity of the DYS14 PCR assay was found to be higher than that of the SRY assay for the detection of fetal DNA when its presence was at a minimum. The DYS14 assay is an improved method for quantifying male fetal DNA in circulating maternal blood in the primate model.

Women's Experiences of Publicly Funded Non-Invasive Prenatal Testing in Ontario, Canada: Considerations for Health Technology Policy-Making.

Non-invasive prenatal testing (NIPT) via fetal DNA in maternal blood has been publicly funded in Ontario, Canada, for high-risk women since 2014. We solicited women's experiences and values related to this new health technology to describe how this test is currently being used in Ontario and to provide information about patient priorities to inform future policy decisions about the use of NIPT. Guided by constructivist grounded theory methodology, we interviewed 38 women who had diverse personal experiences with NIPT. Participants' accounts of their values for decision making about NIPT heavily relied on three mutually modulating factors: timing, accuracy, and risk. The values expressed by women conflict with the way that publicly funded NIPT has typically been implemented in Ontario. We offer recommendations for how NIPT might be integrated into prenatal care pathways in a way more consistent with women's values.

Non-invasive prenatal diagnosis of fetal RhD by using free fetal DNA

Anti-D immunoglobulin is applied to all pregnant women having RhD incompatibility to prevent hemolytic disease of the newborn. The aim of this study is to determine fetal RhD status in the Rh incompatible pregnancies with an non-invasive technique; free fetal DNA isolation from maternal circulation. In the case of Rh incompatibility especially with a history of previous fetal anemia, it can be beneficial to know Rh status antenatally in terms of monitoring fetuses with Rh positive [RhD(+)] status consciously.Total free DNA was isolated in 50 Rh negative [RhD(-)] pregnant women, who had RhD alloimmunisation with their husbands. The gene in isolated DNA was investigated with TagMan prob and real time PCR by using primers belonging to exon 7 of the RhD gene.The authors analyzed 50 RhD(-) women by using quantitative real time PCR technique. Five of them were RhD(-) and the rest of them were found to be RhD(+). After birth one of the infants who were analyzed as RhD(+) were found to be RhD(-).The detection of fetal RhD status by using a non-invasive method from maternal circulation was found to be possible. Assessing fetal RhD status non-invasively by using free fetal DNA in maternal blood will be cost-efficient, avoiding unnecessary indirect Coombs test and unnecessary Rhogam applications that is used in RH incompatible pregnancies. This study will throw a fresh light on prenatal diagnosis.

Incorporación del estudio de ADN fetal en sangre materna al cribado de cromosomopatías

SUMMARY Aims: To evaluate the effectiveness of first trimester combined screening in the prenatal detection of aneu ploidy after 6 years of implantation in our service and its impact in reducing invasive diagnostic tests. It is proposed to establish a protocol to incorporate the study of fetal DNA in maternal blood from published literature reviews. Methods: The risk of fetal chromosomal anomalies was assessed in 3177 pregnancies with first trimester combined screening between January 2009 and December 2014. The amniocenteses performed were checked against those of the previous 5 years. Results: The detection rate of screening for trisomy 21 was 94.4% and the false-positive rate was 6.4%. In 2005 there were 194 amniocenteses. In 2013, 5 years after the introduction of screening, 68 amniocenteses were performed, representing a 70% reduction in invasive procedures. Conclusions: First trimester combined screening has shown a higher detection rate for trisomy 21 that the second trimester screening and/or maternal age, and has substantially reduced the use of invasive prenatal diagnostics procedures. In the coming years, the incorporation of the study of fetal DNA improve the detection of aneuploidys with a drastic reduction of invasive tests so that, the implementation of new protocols is necessary.

Overview of the impact of noninvasive prenatal testing on diagnostic procedures.

Noninvasive prenatal testing (NIPT) has had a profound influence in the field of prenatal diagnosis since the 1997 discovery of cell-free fetal DNA in maternal blood. Research has progressed rapidly, with clinical data supporting laboratory studies showing that NIPT is highly sensitive and specific for fetal aneuploidy, resulting in marked uptake in the high-risk patient population. The superior accuracy of NIPT compared with conventional screening methods has led to significant decreases in the number of invasive diagnostic procedures, in addition to a concomitant decrease in the number of procedure-related fetal losses. Yet, NIPT has been described as a 'disruptive innovation' due to the considerable changes the technology has commanded on current prenatal screening and diagnostic practices. This review summarizes both institutional and global experience with NIPT uptake, its effect on reducing diagnostic invasive procedures, and the unique challenges that reduced procedural volume may have on physician and trainee proficiency, cytogenetic laboratories, and neonatal outcome.

An Overview on Prenatal Screening for Chromosomal Aberrations.

This article is a review of current and emerging methods used for prenatal detection of chromosomal aneuploidies. Chromosomal anomalies in the developing fetus can occur in any pregnancy and lead to death prior to or shortly after birth or to costly lifelong disabilities. Early detection of fetal chromosomal aneuploidies, an atypical number of certain chromosomes, can help parents evaluate their pregnancy options. Current diagnostic methods include maternal serum sampling or nuchal translucency testing, which are minimally invasive diagnostics, but lack sensitivity and specificity. The gold standard, karyotyping, requires amniocentesis or chorionic villus sampling, which are highly invasive and can cause abortions. In addition, many of these methods have long turnaround times, which can cause anxiety in mothers. Next-generation sequencing of fetal DNA in maternal blood enables minimally invasive, sensitive, and reasonably rapid analysis of fetal chromosomal anomalies and can be of clinical utility to parents. This review covers traditional methods and next-generation sequencing techniques for diagnosing aneuploidies in terms of clinical utility, technological characteristics, and market potential.

An endogenous retrovirus involved in placental morphogenesis in Bovinae: diversity of mammalian placentas and endogenous retroviruses

An endogenous retrovirus involved in placental morphogenesis in Bovinae: diversity of mammalian placentas and endogenous retroviruses

Exome sequencing for gene discovery in lethal fetal disorders--harnessing the value of extreme phenotypes.

Massively parallel sequencing has revolutionized our understanding of Mendelian disorders, and many novel genes have been discovered to cause disease phenotypes when mutant. At the same time, next-generation sequencing approaches have enabled non-invasive prenatal testing of free fetal DNA in maternal blood. However, little attention has been paid to using whole exome and genome sequencing strategies for gene identification in fetal disorders that are lethal in utero, because they can appear to be sporadic and Mendelian inheritance may be missed. We present challenges and advantages of applying next-generation sequencing approaches to gene discovery in fetal malformation phenotypes and review recent successful discovery approaches. We discuss the implication and significance of recessive inheritance and cross-species phenotyping in fetal lethal conditions. Whole exome sequencing can be used in individual families with undiagnosed lethal congenital anomaly syndromes to discover causal mutations, provided that prior to data analysis, the fetal phenotype can be correlated to a particular developmental pathway in embryogenesis. Cross-species phenotyping allows providing further evidence for causality of discovered variants in genes involved in those extremely rare phenotypes and will increase our knowledge about normal and abnormal human developmental processes. Ultimately, families will benefit from the option of early prenatal diagnosis.

Non-Invasive Prenatal Diagnosis in the Management of Preimplantation Genetic Diagnosis Pregnancies.

Prenatal diagnosis (PD) is recommended in pregnancies after a Preimplantation Genetic Diagnosis (PGD). However, conventional PD entails a risk of fetal loss which makes PGD patients reluctant to undergo obstetric invasive procedures. The presence of circulating fetal DNA in maternal blood allows performing a non-invasive prenatal diagnosis (NIPD) without risk for the pregnancy outcome. This work shows the introduction of NIPD for confirmation of PGD results in eight pregnancies. In those pregnancies referred to PGD for an X-linked disorder (six out of eight), fetal sex determination in maternal blood was performed to confirm fetal sex. One pregnancy referred to PGD for Marfan syndrome and one referred for Huntington disease (HD) were also analyzed. In seven out of eight cases, PGD results were confirmed by NIPD in maternal blood. No results were obtained in the HD pregnancy. NIPD in PGD pregnancies can be a reliable alternative for couples that after a long process feel reluctant to undergo PD due to the risk of pregnancy loss.

Non-Invasive Prenatal Testing: Ethics and Policy Considerations

New technologies analyzing fetal DNA in maternal blood have led to the wide commercial availability of non-invasive prenatal testing (NIPT). We present here for clinicians the ethical and policy issues related to an emerging practice option. Although NIPT presents opportunities for pregnant women, particularly women who are at increased risk of having a baby with an abnormality or who are otherwise likely to access invasive prenatal testing, NIPT brings significant ethics and policy challenges. The ethical issues include multiple aspects of informed decision-making, such as access to counselling about the possible results of the test in advance of making a decision about participation in NIPT. Policy considerations include issues related to offering and promoting a privately available medical strategy in publicly funded institutions. Ethics and policy considerations merge in NIPT with regard to sex selection and support for persons living with disabilities. RésuméLes nouvelles techniques qui font appel à l’analyse de l’ADN fœtal dans le sang maternel ont mené à l’élargissement de la disponibilité commerciale du dépistage prénatal non effractif (DPNE). Nous présentons ici, à l’intention des cliniciens, les questions d’éthique et de politique qui sont associées à une option de pratique émergente. Bien que le DPNE offre des possibilités aux femmes enceintes (et particulièrement aux femmes qui sont exposées à des risques accrus de donner naissance à un enfant présentant une anomalie ou qui sont autrement susceptibles d’avoir recours au dépistage prénatal effractif), il donne lieu à des défis considérables en matière d’éthique et de politique. Les multiples aspects de la prise d’une décision éclairée (comme l’accès à des services de counseling au sujet des résultats possibles du test avant la prise d’une décision quant à la participation au DPNE) font partie des enjeux éthiques. Parmi les considérations en matière de politique, on trouve les questions liées à l’offre et à la promotion, au sein d’établissements subventionnés par l’État, d’une stratégie médicale offerte au privé. Les considérations en matière d’éthique et de politique s’entremêlent dans le cas du DPNE, pour ce qui est des questions relevant de la sélection du sexe et de l’offre d’un soutien aux personnes qui vivent avec des incapacités.

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.

BackgroundNon-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.MethodsWe 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.ResultsAt 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.ConclusionsNIPT 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.

What does next-generation sequencing mean for prenatal diagnosis?

The ability to gain genetic information from the fetus in the mother's blood during pregnancy has been a long desired goal of research in prenatal medicine. The detection of fetal DNA in maternal blood, coupled with the development of the powerful techniques of next-generation sequencing finally transferred this analysis into clinical practice. Following the commercial introduction of noninvasive prenatal testing for aneuploidies, there has been a very strong demand, which has fostered an extreme rapid development and improvement of technology. Publications in this field are so numerous so that it is challenging to keep up with the latest state of the art. Here, we describe the current basic concepts of cell-free DNA-based noninvasive prenatal testing, give an overview of the currently commercially available tests and the chromosomal aberrations that can be identified. We also present current and future concepts for the implementation of cell-free DNA testing into clinical care.

Antenatal Noninvasive DNA Testing: Clinical Experience and Impact

Nearly two decades ago, the discovery of circulating cell-free fetal DNA in maternal blood created a paradigm shift in prenatal testing. Recent advances in DNA sequencing technology have facilitated the rapid translation of DNA-based testing into clinical antenatal care. In this review, we summarize the technical approaches and current clinical applications of noninvasive testing using cell-free DNA in maternal plasma. We discuss the impact of these tests on clinical care, outline proposed integration models, and suggest future directions for the field. The use of cell-free DNA in maternal blood for the detection of fetal rhesus D antigen status, fetal sex, and common whole chromosomal aneuploidies is now well established, although testing for aneuploidy is still considered screening and not diagnostic. Further advances in technology and bioinformatics may see future clinical applications extend to the noninvasive detection of fetal subchromosomal aneuploidy, single gene disorders, and the entire fetal genome.

Cell-free fetal DNA in maternal blood – an update of the method and clinical practice

Cell-free fetal DNA in maternal blood – an update of the method and clinical practice