Analysis Of Fetal DNA Research Articles

Research Article Noninvasive pre-natal diagnosis of sex by maternal cell-free plasma fetal DNA analysis

Research Article Noninvasive pre-natal diagnosis of sex by maternal cell-free plasma fetal DNA analysis

614: Artificial Intelligence and epigenomic analysis of cell-free fetal DNA for Congenital Heart Defect (CHD) detection

614: Artificial Intelligence and epigenomic analysis of cell-free fetal DNA for Congenital Heart Defect (CHD) detection

Role of molecular genetic non-invasive laboratory diagnostics in prevention of RH-conflict pregnancy

The review is devoted to the possibility of non-invasive prenatal determination of the RH-factor of the fetus in early pregnancy based on the analysis of fetal DNA circulating in the peripheral blood of a pregnant woman. Methodical approaches to the isolation and analysis of extracellular fetal DNA are considered. The assessment of medical and economic efficiency of prenatal noninvasive screening of RH-factor of the fetus in RH-negative pregnant women is given. The possibilities, limitations and prospects of using molecular genetic non-invasive laboratory methods to determine the RH-factor of the fetus are discussed.

Cell-free fetal DNA analysis: Translating innovations into clinical practice

Cell-free fetal DNA analysis: Translating innovations into clinical practice

Effectiveness of Prenatal Intervention on the Outcome of Diseases That Have a Postnatal Urological Impact.

We reviewed the literature addressing outcomes of fetal intervention of conditions that require post-natal urological management including lower urinary tract obstruction, hydrometrocolpos, congenital adrenal hyperplasia, and myelomeningocele. Despite several decades of fetal intervention for these conditions, benefits remain elusive in part because of the enormous difficulty of conducting prospective randomized studies. In this review, we reached the following conclusions: Prenatal intervention in lower urinary tract obstruction (LUTO) improves survival in the worst cases by improving pulmonary function and it may be advantageous for renal function, particularly in patients with adverse urine parameters.Prenatal treatment of female fetuses at risk of having congenital adrenal hyperplasia (CAH) reduces virilization. Non-invasive fetal DNA analysis allows earlier diagnosis, reducing the risk of treating with dexamethasone males and non-affected fetuses.Fetal treatment of myelomeningocele (MMC) is gaining momentum. Prospective studies including standardized urologic evaluation will determine if the encouraging results reported by some hold on the long term follow-up.

Targeted Rhesus immunoglobulin for RhD-negative women undergoing an induced abortion: A clinical pilot study.

The aim of this clinical pilot study was to examine the accuracy of noninvasive fetal RHD genotyping in early pregnancy (8+0 to 11+6 weeks) and to clarify whether targeted administration of Rhesus immunoglobulin (RhIg) is possible for women undergoing an induced abortion such that unnecessary injections can be avoided. The study examines the correlation between gestational age and the amount of cell-free fetal DNA in maternal plasma, the fetal fraction of DNA and whether transportation time or body mass index affects these parameters. Fifty-two RhD-negative women undergoing a surgically induced abortion were included. A maternal blood sample was collected prior to the abortion and a tissue sample was collected from the placental part of the abortion material after the intervention. Fetal RhD type was determined by PCR analysis of cell-free fetal DNA extracted from maternal plasma and on DNA from the tissue sample, with the latter providing a reference standard. Copies of RHD/mL were determined on RHD-positive samples and the fetal fraction of DNA was calculated. We demonstrated complete concordance between results from plasma and tissue, with 31 RhD-positive and 21 RhD-negative samples, corresponding to 40% being RhD-negative, specificity 100% [95% confidence interval (CI) 88.8-100] and sensitivity 100% (95% CI 83.9-100). We found no significant correlation between gestational age and the amount or the fraction of cell-free fetal DNA in maternal plasma, nor did we find that transportation time or BMI significantly affected these factors in this setup. Fetal RHD genotyping can be accurately performed from the 8th week of gestation and unnecessary injections of RhIg can be avoided for women undergoing an induced abortion. A larger study is needed to determine a more accurate sensitivity for the analysis early in pregnancy.

Analysis of fetal DNA in maternal plasma with markers designed for forensic DNA mixture resolution

PurposeWith the description of circulating fetal DNA in maternal blood,noninvasive prenatal diagnostics became theoretically possible. As the presenceof background maternal DNA interferes with the detection of fetal DNA,analytical methods require genetic markers capable of distinguishing byquantitative or targeted approaches the minor population of DNA molecules of thefetus. Here we evaluate the feasibility of analyzing fetal DNA with novelDIP-STR genetic markers, designed for the investigation of forensic mixedbiological evidence. MethodsThe DIP-STR molecular approach is based on sequence-specificanalysis of paternally inherited fetal alleles. These sequences are biallelicdeletion/insertion polymorphisms (DIPs) located very close to short tandemrepeat (STR) markers, for combined analysis. In this study, 48 women were testedwith 28 DIP-STRs during the first, second, and third trimester ofpregnancy. ResultsPositive results were obtained across markers, including longer ones(386 base-pairs) and with blood samples collected during early pregnancy, suchas 10 weeks of gestational age. ConclusionThese data show that DIP-STR markers can be used to amplify specificgenomic regions of circulating fetal DNA to obtain targeted genetic information.This method may contribute to developments in noninvasive prenatal paternitytesting and diagnosis of certain genetic diseases.

Prenatal Diagnosis in the Netherlands

Prenatal invasive diagnosis of genetic conditions in the Netherlands is well organised, based on uniform indications and has a sound financial structure. Facilities for fetal karyotyping and DNA analysis are available in the 8 academic centres. Prenatal diagnosis of metabolic diseases is mainly carried out in Rotterdam. Amniocentesis, transcervical and transabdominal chorionic villus sampling are carried out in all centres, including 4 subcentres. The national Working Party on Prenatal Diagnosis started in 1985 and is a useful platform for all obstetricians and geneticists involved in prenatal diagnosis.

Just choice: a Danielsian analysis of the aims and scope of prenatal screening for fetal abnormalities

Developments in Non-Invasive Prenatal Testing (NIPT) and cell-free fetal DNA analysis raise the possibility that antenatal services may soon be able to support couples in non-invasively testing for, and diagnosing, an unprecedented range of genetic disorders and traits coded within their unborn child’s genome. Inevitably, this has prompted debate within the bioethics literature about what screening options should be offered to couples for the purpose of reproductive choice. In relation to this problem, the European Society of Human Genetics (ESHG) and American Society of Human Genetics (ASHG) tentatively recommend that any expansion of this type of screening, as facilitated by NIPT, should be limited to serious congenital and childhood disorders. In support of this recommendation, the ESHG and ASHG cite considerations of distribution justice. Notably, however, an account of justice in the organization and provision of this type of screening which might substantiate this recommendation has yet to be developed. This paper attempts to redress this oversight through an investigation of Norman Daniels’ theory of Just health: meeting health needs fairly. In line with this aim, the paper examines what special moral importance (for Just health) screening for the purpose of reproductive choice might have where concerning serious congenital and childhood disorders in particular. The paper concludes that screening for reproductive choice where concerning serious congenital and childhood disorders may be important for providing women with fair opportunity to protect their health (by either having or not having an affected child).

Non-invasive Prenatal Testing Using Fetal DNA.

Non-invasive prenatal diagnosis (NIPD) is based on fetal DNA analysis starting from a simple peripheral blood sample, thus avoiding risks associated with conventional invasive techniques. During pregnancy, the fetal DNA increases to approximately 3-13% of the total circulating free DNA in maternal plasma. The very low amount of circulating cell-free fetal DNA (ccffDNA) in maternal plasma is a crucial issue, and requires specific and optimized techniques for ccffDNA purification from maternal plasma. In addition, highly sensitive detection approaches are required. In recent years, advanced ccffDNA investigation approaches have allowed the application of non-invasive prenatal testing (NIPT) to determine fetal sex, fetal rhesusD (RhD) genotyping, aneuploidies, micro-deletions and thedetection of paternally inherited monogenic disorders. Finally, complex and innovative technologies such as digital polymerase chain reaction (dPCR) and next-generation sequencing (NGS) (exhibiting higher sensitivity and/or the capability to read the entire fetal genome from maternal plasma DNA) are expected to allow the detection, in the near future, of maternally inherited mutations that cause genetic diseases. The aim of this review is to introduce the principal ccffDNA characteristics and their applications as the basis of current and novel NIPT.

Cell-free Fetal DNA Analysis in Maternal Plasma as Screening Test for Trisomies 21, 18, and 13 in Twin Pregnancy

(Abstracted from Ultrasound Obstet Gynecol 2018;52(3):318–324) Increased use of fertility treatment and advanced maternal age contribute to the increased prevalence of twin pregnancy, which is currently approximately 3% in the United States. Screening for trisomy 21 in twins largely relies on maternal age and nuchal translucency measurement in the first trimester, as well as first- and second-trimester serum screening.

A Novel Next-Generation Sequencing Based Assay for Non-Invasive Prenatal Testing of Sickle Cell Disease without Paternal DNA

Introduction: Over 300,000 infants are born with sickle cell disease (SCD) every year worldwide, including at least 1,000 in the US. Prenatal diagnosis by amniocentesis or chorionic villus sampling is available; but high cost, invasiveness, and risk of miscarriage limit their use. Recently, non-invasive prenatal testing (NIPT) has become commonplace for aneuploidies, including Trisomy 21. These non-invasive tests operate by genetic analysis of the cell-free fetal DNA (cffDNA) present in maternal blood. The safety and accuracy of NIPT have been key drivers for its rapid and widespread adoption. Yet, no NIPT for SCD or other hemoglobinopathies have been commercialized to date, despite the large numbers of patients affected in the US and worldwide. While de novo mutations can only be of fetal origin and can be identified by available next-generation sequencing (NGS) methods, NIPT for recessively inherited disorders is more challenging. This is because a mother who is a carrier for a recessive disorder contributes a high level of background pathogenic DNA molecules. Therefore, a key technical challenge of NIPT for recessive disorders is developing an assay sensitive enough to detect <5% deviation from 50% allele fraction. To overcome this challenge, we have developed and optimized an NIPT for SCD by assessing the relative mutation dosage of fetal SCD and beta-thalassemia DNA through a novel molecular counting strategy using NGS.Objectives: The primary objective of this study is to evaluate the performance of a novel NIPT for sickle cell disease.Methods: The SCD NIPT assay and associated custom bioinformatics analysis were performed on cfDNA obtained from a training cohort of non-pregnant compound heterozygotes for SCD. The SCD NIPT assay was then performed on a validation cohort of pregnant women with either SCD or sickle cell trait (SCT). The accuracy of the SCD NIPT was evaluated by comparison with newborn screening results.Results: Non-pregnant individuals with genotype HbSE, HbSC, or HbS/beta-thalassemia were included as a training cohort to establish the precision and accuracy of the assay for measuring HbS allele fraction from cfDNA. As expected, the HbS allele fraction in these individuals was 0.500 (standard deviation = 0.011, n = 26), and there was no detectable fetal fraction in these samples. Both training and validation cohort results matched the theoretical limit of detection set by the number of cell-free HBB DNA molecules in plasma. The precision and accuracy of the HBB assay on cfDNA were then used in conjunction with >1000 pre-clinical samples (mixtures of sheared SCT and SCD genomic DNA) to determine analytical sensitivity >98% and specificity >99%, even in the absence of paternal DNA.Conclusion: We have developed an assay for non-invasive prenatal testing of sickle cell disease. The results obtained to date indicate that the assay reliably detects fetal SCD when the fetal fraction is as low as 5%, the same limit as aneuploidy NIPT. A fetus with SCD has already been identified, and follow-up is ongoing with >20 pregnancies. Since the HBB NIPT is highly targeted, sequencing cost is <$30 per sample. The ability to ascertain fetal SCD status based only on maternal blood will be valuable in clinical settings where the father is unavailable or sample collection would be inconvenient or time-consuming. Several Phase I/II and Phase III trials for curing SCD or beta-thalassemia using autologous gene-editing of stem cells are currently in progress. SCD NIPT could be particularly useful for deciding to bank umbilical cord blood as a source of stem cells for future gene-editing cures. DisclosuresTsao:BillionToOne: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Atay:BillionToOne: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Benefits, challenges and ethical principles associated with implementing noninvasive prenatal testing: a Delphi study.

Noninvasive prenatal testing is a recent technology that provides some genetic information about the fetus through the analysis of cell-free fetal DNA circulating in maternal blood. We aimed to identify the benefits, challenges and guiding ethical principles most relevant to the clinical integration of noninvasive prenatal testing in Canada, according to experts throughout the country. We conducted a 3-round Delphi study involving Canadian experts of contemporary discussions about the ethical and societal implications of prenatal testing and genomic technologies. In round 1, we asked participants to identify clinical benefits and challenges related to the implementation of noninvasive prenatal testing in Canada, and the ethical principles they think should guide it. In round 2, we asked participants to select the most important elements stated by their peers. In round 3, participants were informed of the aggregated results from round 2, and invited to revise or confirm their selection. Round 1 had a participation rate of 20.2%, and involved 61 participants. Subsequent rounds 2 and 3 had retention rates of 95.1% (n = 58) and 84.5% (n = 49), respectively. Through these discussions, we identified 3 lists of benefits (n = 10), challenges (n = 27), and ethical principles (n = 16) prioritized by Canadian experts as being most relevant to the implementation of noninvasive prenatal testing in Canada. Although multiple and diverse potential issues were identified, Canadian experts agreed on 2 sets of requirements for the responsible implementation of noninvasive prenatal testing in Canada. Interdisciplinary appraisals may be instrumental to responsible policy-making related to the implementation of noninvasive prenatal testing in Canada.

Trends in the use of prenatal testing services for fetal aneuploidy in Ontario: a descriptive study.

In 2014, Ontario augmented its publicly funded multiple-marker screening program for prenatal aneuploidy by incorporating cell-free fetal DNA (cffDNA) analysis for high-risk pregnancies. We assessed trends in the use of multiple-marker screening, cffDNA screening and prenatal diagnostic testing before and after implementation of public funding. We conducted a descriptive study based on data from the Better Outcomes Registry & Network (BORN) Ontario. The study population included all pregnant women in Ontario with a singleton pregnancy and an expected date of delivery between July 1, 2012, and Mar. 31, 2016, with pregnancy data captured in BORN. Pregnancy losses and terminations before 20 weeks' gestation not captured in BORN were excluded. We generated descriptive statistics to show trends and regional variations in use. The study sample included 534 210 singleton pregnancies. After cffDNA screening was funded for specific indications, uptake of multiple-marker screening increased slightly, from 66.5% to 68.1% (p < 0.001). Uptake of cffDNA screening among women with a positive multiple-marker screening result increased substantially, from 3.2% to 48.8% (p < 0.001). In contrast, the rate of prenatal diagnostic testing in this group decreased from 54.8% to 30.8% (p < 0.001). Although women aged 40 years or older are eligible for primary cffDNA screening, only a small decrease in the use of multiple-marker screening was observed in this group. The greatest use of cffDNA screening and greatest decline in prenatal diagnostic testing were seen in women with a level of risk for trisomy 21 of 1:101-1:200 based on multiple-marker screening. After public funding of cffDNA screening was implemented in Ontario, there was a significant increase in cffDNA screening and a significant decrease in prenatal diagnostic testing among women with a positive multiple-marker screening result. These changing patterns show the significant impact of public policy and funding decisions on women's choices regarding prenatal testing.

Evaluation of Noninvasive Prenatal Testing (NIPT) guidelines using the AGREE II instrument

Objective: The rapid increase of cell-free fetal DNA analysis for Down syndrome screening requires evidence-based clinical practice guidelines for noninvasive prenatal testing (NIPT). Several studies show that the quality of many guidelines is low and there are still many health areas where this quality is not systematically evaluated. Given the absence of research, in the NIPT field, we used an internationally validated tool to evaluate a set of three NIPT practice guidelines and to look at dimensions that can be improved.Methods: Four appraisers, experts in prenatal screening, evaluated three main NIPT guidelines published in the last 2 years using the AGREE II (Appraisal of Guidelines for Research and Evaluation II), a tool specifically designed for guideline quality appraisal.Results: Guidelines scored higher in domains related with scope, purpose, and clarity of presentation, and lower in stakeholder involvement and rigor of development. Intradomain items evaluation showed asymmetries between guidelines. The UK-NSC was the guideline with the best scores.Discussion: Several areas of NIPT guidelines, such as stakeholders involvement, selection of supporting evidence, external reviews, updating processes, and competing interests disclosure, can be improved. Appraisers recommend modifications to all NIPT guidelines that can lead to substantial improvements in their methodological quality and subsequently make a contribution to prenatal screening improvement.

A Broad Test Based on Fluorescent-Multiplex PCR for Noninvasive Prenatal Diagnosis of Cystic Fibrosis

Background: Analysis of cell-free fetal DNA in maternal plasma is very promising for early diagnosis of monogenic diseases. However, it has been limited by the need to set up patient- or disease-specific custom-made approaches. Here we propose a universal test based on fluorescent multiplex PCR and size fragment analysis for an indirect diagnosis of cystic fibrosis (CF). Methods: The test, based on haplotyping, includes nine intra- and extragenic short tandem repeats of the CFTR locus, the coamplification of p.Phe508del (the most frequent mutation in CF patients worldwide), and a specific SRY sequence. The assay is able to determine the inherited paternal allele. Results: Our simple approach was successfully applied to 30 couples and provided clear results from the maternal plasma. The mean rate of informative markers was sufficient to propose it for use in indirect diagnosis. Conclusions: This noninvasive prenatal diagnosis test, focused on indirect diagnosis of CF, offers many advantages over current methods: it is simple, rapid, and cost-effective. It allows for the testing of a large number of couples with high risk of CF, whatever the familial mutation of the CFTR gene. It provides an alternative method to reduce the number of invasive tests.

Noninvasive fetal sex determination by analysis of cell-free fetal DNA in maternal capillary blood obtained by fingertip puncture.

What's already known about this topic? Noninvasive fetal sex determination searches for Y‐chromosome sequences in maternal plasma isolated from venous blood drawn by venipuncture to indicate fetal sex in the first trimester of pregnancy. Capillary blood collection is much less invasive than venipuncture, thereby offering the possibility of sampling in geographical areas where there is no phlebotomist present. What does this study add? Fetal DNA is present in maternal capillary blood allowing for noninvasive fetal sex determination. Exogenous male DNA may be present, and detectable, on the fingertips of pregnant women. The elimination of the exogenous male DNA is critical for a reliable fetal sex determination using maternal capillary blood. A diluted buffered sodium hypochlorite (0.5%‐1%) can be used to eliminate exogenous male DNA from the fingertips of pregnant women.

Complex Analysis of Total and Fetal DNA and Cytokines in Blood Plasma of Pregnant Women with Preeclampsia.

We performed a complex analysis of total and fetal extracellular DNA, 8 cytokines (IL-2, IL-4, IL-6, IL-8, IL-10, granulocyte-macrophage CSF, IFNγ, and TNFα) in blood plasma obtained from women with preeclampsia prior to labor onset. Total (sensitivity 89.47%, specificity 93.75%) and fetal extracellular DNA (sensitivity 73.68%, specificity 87.5%) were the most accurate parameters determining preeclampsia. We revealed a high correlation (p=3×10-6) between total and fetal extracellular DNA levels in the group of preeclampsia. Preeclampsia significantly increased the levels of macrophage factors IL-10 and IL-6. These cytokines significantly correlated with the levels of total and fetal extracellular DNA in the preeclampsia group. In the control group, such correlations were not observed. These findings obtained suggest that preeclampsia develops upon increased macrophage activity, leading to destruction of the placenta trophoblast cells.

Positive view and increased likely uptake of follow-up testing with analysis of cell-free fetal DNA as alternative to invasive testing among Danish pregnant women.

The aim of this study was to investigate the attitude (view, likely uptake and preferred strategy) towards cell-free fetal DNA (cfDNA) testing among pregnant women before a first-trimester risk assessment for trisomy 21 (unselected women) and after obtaining a high risk. Unselected and high-risk women attending first-trimester screening (Rigshospitalet, Copenhagen University Hospital) were invited to fill out the questionnaire Antenatal testing for Down syndrome as an online survey. The survey included 203 unselected and 50 high-risk women (response rates of 74.8% and 84.7%, respectively). Nearly all considered cfDNA testing a positive development in antenatal care, and 97.2% would like it to be offered. Offering cfDNA testing as an alternative to invasive testing would increase the uptake of follow-up testing compared with invasive testing alone (98.8% vs. 90.7%, p<0.001). Women who would only accept follow up by cfDNA testing were more likely to continue an affected pregnancy (30.0% vs. 3.6%, p<0.001) or have doubts about termination (50.0% vs. 32.1%, p<0.001). Offering cfDNA testing would likely increase the uptake of follow-up testing without a corresponding rise in the termination rate of affected fetuses as some women test for information only. However, both unselected and high-risk women had overwhelmingly positive views underlining attention to avoid routinization.

CfDNA screening and diagnosis of monogenic disorders - where are we heading?

Cell‐free fetal DNA analysis for non‐invasive prenatal screening of fetal chromosomal aneuploidy has been widely adopted for clinical use. Fetal monogenic diseases have also been shown to be amenable to non‐invasive detection by maternal plasma DNA analysis. A number of recent technological developments in this area has increased the level of clinical interest, particularly as one approach does not require customized reagents per mutation. The mutational status of the fetus can be assessed by determining which parental haplotype that fetus has inherited based on the detection of haplotype‐associated SNP alleles in maternal plasma. Such relative haplotype dosage analysis requires the input of the parental haplotype information for interpretation of the fetal inheritance pattern from the maternal plasma DNA data. The parental haplotype information can be obtained by direct means, reducing the need to infer haplotypes using DNA from other family members. The technique also allows the assessment of complex mutations and has multiplexing capabilities where a number of genes and mutations can be assessed at the same time. These advantages allow non‐invasive prenatal diagnosis of fetal monogenic diseases to be much more scalable. These applications may drive the next wave of clinical adoption of cell‐free fetal DNA testing. © 2018 The Authors Prenatal Diagnosis Published by John Wiley & Sons Ltd