Fetal DNA Testing Research Articles

Genetic diagnosis and non-invasive prenatal testing of a fetus with Prader-Willi/Angelman syndrome

To explore the genetic basis for a fetus featuring growth restriction and validate the effectiveness of a novel noninvasive prenatal testing (NIPT) technique for the detection of chromosomal microdeletions. Next-generation sequencing(NGS) and fluorescence in situ hybridization(FISH) were used to analyze the DNA of the fetus. Conventional G-banding was used to analyze the karyotypes of the fetus and its parents. High-throughput sequencing was used to analyze free fetal DNA. NGS analysis has revealed a 4.88 Mb deletion at 15q11.2-q13.1 region in the fetus, which has a 99% overlap with the critical region of Prader-Willi syndrome (Type 2) and Angelman syndrome (Type 2) and encompassed critical genes including SNRPN and UBE3A. NIPT also revealed a 4.6 Mb deletion at 15q12, which was consistent with the results of fetal cord blood and amniotic DNA testing. FISH assay has confirmed the result of NGS. By karyotying, all subjects showed a normal karyotypes at a level of 320~400 bands. It is quite necessary to carry out genetic testing on fetuses showing growth restriction. NIPT for fetal chromosomal microdeletions/microduplication syndromes is highly accurate for the diagnosis of Prader-Willi/Angelman syndrome.

Improving patient care in monogenic diabetes through research and education

AbstractThe 2019 Arnold Bloom lecture reviewed the progress in monogenic diabetes since the start of the service in Exeter in 1995 to present day.The impact of a correct molecular genetic diagnosis has been profound for many patients who have been able to stop insulin injections even after many years on this treatment. However, there remain many patients who are still not correctly diagnosed and long delays between initial diabetes diagnosis and correct genetic diagnosis remain. Raising awareness of monogenic diabetes across the UK has been aided by a national network of genetic diabetes nurses (GDNs). GDNs educate health care professionals about genetic forms of diabetes and are able to provide advice to patients and their families following a positive genetic diagnosis.Challenges in the management of monogenic diabetes still remain. Systematic approaches are being instigated to ensure correct molecular genetic diagnosis is made as close to the initial diabetes diagnosis as possible, and research is being undertaken to identify what support is needed for those receiving ‘unexpected’ genetic results. Management of monogenic diabetes pregnancy can be complex as many women with the most common forms of monogenic diabetes are best treated with sulphonylureas outside pregnancy and fetal genotype can affect birthweight so there are a number of issues to consider. Future management of pregnancy in these cases may be aided by use of cell free fetal DNA testing. Further information on monogenic diabetes can be found at www.diabetesgenes.org. Copyright © 2019 John Wiley & Sons.

Impact of cell-free fetal DNA on invasive prenatal diagnostic tests in a real-world public setting.

Objective To evaluate the impact of cell-free fetal DNA (cfDNA) test on the number of invasive tests carried out in a public hospital that does not include this test in its services. Methods This was a retrospective cohort study in singleton pregnancies with a high risk (>1:270) on the first-trimester screening for aneuploidies. The options of performing an invasive test or a cfDNA test were explained to all women, the latter being especially recommended to those with a 1:50-1:270 risk (Group 1). If the risk was >1:50 (Group 2), or nuchal translucency (NT) was >99th percentile or there were major malformations (Group 3), invasive test was recommended. Results A total of 755 of 14,398 (5.2%) cases had a high-risk first-trimester screening, of whom 46 cases were excluded due to incomplete follow-up. In the remaining 709 cases, the percentage of aneuploidies was 9.9% (70 cases) and 110 opted for a cfDNA test (15.5%). There were two true-positive results of cfDNA (one in Group 2 and another in Group 3). In Group 1, 67.4% [95% confidence interval (CI) 60.0%-72.1%, P < 0.01] fewer invasive procedures were performed in those who opted for a cfDNA test, without having false negatives. Conclusion Pregnant women with a 1:50-1:270 risk who opt for cfDNA save two out of three invasive tests, without affecting the aneuploidy detection rate.

High-throughput, non-invasive prenatal testing for fetal rhesus D status in RhD-negative women: a systematic review and meta-analysis

BackgroundHigh-throughput non-invasive prenatal testing (NIPT) for fetal Rhesus D (RhD) status could avoid unnecessary treatment with anti-D immunoglobulin for RhD-negative women found to be carrying an RhD-negative fetus. We aimed to assess the diagnostic accuracy of high-throughput NIPT for fetal RhD status in RhD-negative women not known to be sensitized to the RhD antigen, by performing a systematic review and meta-analysis.MethodsProspective cohort studies of high-throughput NIPT used to determine fetal RhD status were included. The eligible population were pregnant women who were RhD negative and not known to be sensitized to RhD antigen. The index test was high-throughput, NIPT cell-free fetal DNA tests of maternal plasma used to determine fetal RhD status. The reference standard considered was serologic cord blood testing at birth. Databases including MEDLINE, EMBASE, and Science Citation Index were searched up to February 2016.Two reviewers independently screened titles and abstracts and assessed full texts identified as potentially relevant. Risk of bias was assessed using QUADAS-2. The bivariate and hierarchical summary receiver-operating characteristic (HSROC) models were fitted to calculate summary estimates of sensitivity, specificity, false positive and false negative rates, and the associated 95% confidence intervals (CIs).ResultsA total of 3921 references records were identified through electronic searches. Eight studies were included in the systematic review. Six studies were judged to be at low risk of bias. The HSROC models demonstrated high diagnostic performance of high-throughput NIPT testing for women tested at or after 11 weeks gestation. In the primary analysis for diagnostic accuracy, women with an inconclusive test result were treated as having tested positive. The false negative rate (incorrectly classed as RhD negative) was 0.34% (95% CI 0.15 to 0.76) and the false positive rate (incorrectly classed as RhD positive) was 3.86% (95% CI 2.54 to 5.82). There was limited evidence for non-white women and multiple pregnancies.ConclusionsHigh-throughput NIPT is sufficiently accurate to detect fetal RhD status in RhD-negative women and would considerably reduce unnecessary treatment with routine anti-D immunoglobulin. The applicability of these findings to non-white women and women with multiple pregnancies is uncertain.

Is there a future for cell-free fetal dna tests in screening for preeclampsia?

CffDNA screening is a powerful diagnostic tool in the prenatal diagnosis algorithm for chromosomal abnormalities. With detailed ultrasound examination as the mainstay of first-trimester risk assessment, cffDNA has been shown to be superior to first-trimester combined screening (FTCS) in false-positive rates for trisomy 21 detection. In light of the growing interest in cffDNA testing and the possibility of it replacing first-trimester biochemistry, we decided to investigate the usefulness of cffDNA tests in early-pregnancy risk assessment for preeclampsia (PE). The aim of this review paper was to evaluate clinical application of first-trimester cfDNA in predicting PE, as well as to investigate its possible use in first-trimester PE screening enhancement, also in cases where biochemistry is not performed.

A contingent model for cell-free DNA testing to detect fetal aneuploidy after first trimester combined screening.

ObjectiveTo assess the results of the first trimester combined test to design a prenatal protocol for the introduction of the cell-free fetal DNA test as a contingent screening model.MethodAn observational retrospective study in 12,327 singleton pregnancies to analyze the results of the combined first trimester screening, the nuchal translucency ≥97.5 percentile, their cytogenetic results and birth outcomes.ResultsA total of 533 (4.3%) pregnant women had a risk in combined first trimester screening above 1/300. In this group, sixty nine had an unbalanced karyotype. The abnormal/normal karyotype ratio was 1/28 in pregnant women with intermediate risk (1/51-1/300) for trisomy 21 and trisomy 18, 1/58 with intermediate risk just for trisomy 21 and 1/37 with intermediate risk just for trisomy 18. A 19.8% of the unbalanced karyotypes had chromosomal abnormalities other than trisomies 21, 18 and 13. Two false negatives cases at first trimester combined screening presented a nuchal translucency ≥ p97.5th.ConclusionWe propose the introduction of the cell-free fetal DNA test when the risk of first trimester combined screening is intermediate (1/51–1/300) and when nuchal translucency is ≥ p97.5th with a low risk in the combined screening. This policy would allow us to continue to detect uncommon chromosomal abnormalities.

Autoimmune disorders but not heparin are associated with cell-free fetal DNA test failure

BackgroundRecent studies have suggested a possible association between heparin treatment at the time of cell-free DNA (cfDNA) testing and a non-reportable result. However, these studies lack of proper methodology and had a low level of proof to firmly incriminate heparin. Our objective was to investigate further the relationship between heparin treatment and cfDNA test results.MethodsTwo complementary approaches were used for the demonstration. First, we conducted a retrospective analysis of a cohort of patients with a singleton pregnancy, screened for aneuploidies by using cfDNA, but with a non-reportable cfDNA result. We included patients between 2013 and 2016 including the patients from the DEPOSA study as controls. CfDNA testing was performed by massive parallel sequencing by using a whole-genome approach. A multiple logistic regression was used to account for the influence of the variables included. Second, we performed in vitro experiments on mimic samples containing increased concentrations of heparin.ResultsOf 9867 singleton pregnancies tested during the inclusion period, 58 (0.59%) had a non-reportable result and were compared to 295 control patients. Fifteen (25.9%) and 20 (6.8%) patients were treated with heparin in the group with a non-reportable cfDNA result and with a successful assay, respectively. In multivariable analysis, an increased calculated risk at the first-trimester combined screening (OR 28.8 CI 9.76–85.15, p < 0.001), maternal weight (OR 1.03, CI 1.01–1.06, p = 0.01), and the presence of an autoimmune disease (OR 10.38, CI 1.62–66.53, p = 0.01) were the only characteristics associated with a non-reportable result. In vitro experiments showed that heparin had no impact on fetal fraction measurement or the final result, no matter what the dose tested.ConclusionsTreatment by heparin had no impact on cfDNA screening test for aneuploidies, while the presence of an autoimmune disorder is an independent predictor of a non-reportable result.

Isolated Echogenic Intracardiac Foci: A Comparison of Aneuploidy Screening Tests [28P

INTRODUCTION: The 2014 Fetal Imaging Workshop endorses obtaining a quad screen or cell-free fetal DNA (cfDNA) test in fetuses identified to have an isolated echogenic intracardiac foci (EIF) without prior aneuploidy screening. Our goal was to compare estimated outcomes and costs of the recommendation. We additionally hoped to gain information to assist providers in offering patient specific recommendations. METHODS: A decision-analytic model was constructed using TreeAge software and probabilities derived from the literature. The model compared use of quad testing to cfDNA in women with an isolated EIF identified on screening ultrasound, with T21 sensitivities assumed at 81% and 99% respectively. Baseline risk of T21 was assumed at 1/400. Positive likelihood ratio for T21 with an isolated EIF, was assumed to be 1.5. Outcomes of interest included T21 diagnosis, procedure related loss, termination, spontaneous abortion, and T21 live births. Strategies were compared to generate an incremental cost-effectiveness ratio, with a $100,000/QALY threshold, and applied to a theoretical cohort of 100,000. RESULTS: When compared to aneuploidy screening using quad testing, the strategy that utilized cfDNA after detection of an isolated EIF resulted in 47 fewer live T21 births and 85 additional QALYs with cost savings of $525,964 per 100,000 pregnancies; a dominant strategy in cost-effectiveness analysis. CONCLUSION: In a population with an isolated EIF identified on screening US without prior aneuploidy screening it is cost effective to offer cfDNA as compared to a quad screening test. Despite the higher cost of cfDNA, these results likely stem from the increased sensitivity and decreased false positive rate.

Cell-free fetal DNA testing in singleton IVF conceptions.

Are fetal fraction, test failure rate and positive predictive value (PPV) of cell-free fetal DNA (cffDNA) testing different in singleton IVF conceptions compared to spontaneous conceptions? Fetal fraction is significantly lower; test failure rate is higher and PPV of cffDNA testing is lower in singleton pregnancies conceived by IVF than those conceived spontaneously. cffDNA testing, which analyses circulating cffDNA in maternal blood, has very high accuracy for detection of trisomy 21 in the general obstetric population. Focused and conclusive evidence regarding the test characteristics of cffDNA testing in IVF conceived pregnancies is lacking. This was a retrospective cohort study including spontaneously and IVF conceived singleton pregnancies collected consecutively between April 2013 and November 2016. A total of 4633 spontaneously conceived and 992 IVF pregnancies were included. The study was performed at an obstetric and gynecological ultrasound clinic in Melbourne, Australia. Participants had screening for trisomies 21, 18 and 13, as well as sex chromosome aneuploidies (SCA) performed with cffDNA testing after 10 weeks' gestation. Multivariate regression analysis was used to determine significant predictors of logarithmically transformed fetal fraction and test failure. Comparison of test characteristics between study groups was performed adopting a significance level of 5%. Median fetal fraction was lower (10.3% [interquartile range (IQR), 7.7-13.5] versus 11.9% [IQR, 9.1-15.0]; P = 0.005), test failure rate was higher (5.2 versus 2.2%; P < 0.001) and positive predictive value (PPV) for trisomies 18, 13 and SCA was poorer in IVF pregnancies compared to those spontaneously conceived. Multivariate linear regression analysis demonstrated that IVF conception, increased BMI, earlier gestational age and South and East Asian ethnicities were independent predictors of lower fetal fraction. Multiple logistic regression analysis found IVF conception and increased BMI to be independently associated with test failure. PPV was high for trisomy 21 in IVF conception (100.0%), but was lower for other trisomies when compared with the non-IVF population. IVF details were unascertainable for 210 cases, as the information was not available through our data collection points. Inability to karyotype some cases at high-risk for SCA, due to patients' choice, and the occurrence of miscarriages and terminations, resulted in the exclusion of high-risk cases when calculating PPV. Pregnancy outcomes were not available in low-risk pregnancies and negative predictive values could not be calculated. The limitations revealed by this work should be taken into account during pre-test counseling in pregnant women who conceive by IVF. No external source of financial support was provided for this research. The authors report no conflicts of interest.

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.

Performance of prenatal screening by non-invasive cell-free fetal DNA testing for women with various indications

OBJECTIVE To assess the performance of non-invasive prenatal testing (NIPT) based on massive parallel sequencing. METHODS A total of 10 275 maternal blood samples were collected. Fetal chromosomal aneuploides were subjected to low coverage whole genome sequencing. Patients with high risks received further prenatal diagnosis. The outcome of all patients were followed up. RESULTS High-throughput sequencing detected 72 pregnancies with fetal autosomal chromosomal aneuploidy, including 57 cases of trisomy 21, 14 cases of trisomy 18, and 1 case of trisomy 13. The positive predictive value for trisomies 21 and 18 were 98.25% and 91.67%, respectively. Comparing its performance in intermediate or high risk pregnancies, advanced maternal age pregnancies and volunteering to test pregnancies, the positive predictive value were 100%, 95%, 90% and 50%, respectively. The follow up result was only 1 case of 21 trisomy false negative with high risk. For the 56 cases of trisomy 21, the high risk group accounted for 55%, advanced maternal age accounted for 29%, the intermediate risk referred to 14%, the volunteering to test group accounted for 2%. CONCLUSION The performance of NIPT for trisomies 21, 18 and 13 was satisfactory. The method can be used for women with advanced gestational age. NIPT has offered an ideal secondary screening method for those with an intermediate or high risk, and can reduce the rate of birth defects.

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

Controversies in Pregnancy Management after Prenatal Diagnosis of a Twin Pregnancy Discordant for Trisomy 21 Diagnosed by Cell-Free Fetal DNA Testing

Background: Current preliminary data is advocating cfDNA testing in twin pregnancies since both increasing use of ART and higher maternal ages have raised the incidence of (discordant) aneuploidies. Procedures and findings:This report is raising ethical implications deriving from a twin pregnancy discordant for trisomy 21 conceived from egg donation and diagnosed by cfDNA testing after low risk conventional first-trimester screening. For More Information : https://symbiosisonlinepublishing.com/

Cell-free fetal DNA testing in prenatal diagnosis: Recommendations of the Polish Gynecological Society and the Polish Human Genetics Society

This paper contains a joint position of the Polish Gynecological Society and Polish Human Genetics Society on the cell-free fetal DNA testing in prenatal diagnosis. We present situations where the cell-free fetal DNA testing should be applied and cases in which performing of the test is not useful. We indicate what diagnostic steps should be performed before the test and how the test results should be interpreted and followed.

Massively Parallel Sequencing (MPS) of Cell-Free Fetal DNA (cffDNA) for Trisomies 21, 18, and 13 in Twin Pregnancies.

Massively parallel sequencing (MPS) technology has become increasingly available and has been widely used to screen for trisomies 21, 18, and 13 in singleton pregnancies. This study assessed the performance of MPS testing of cell-free fetal DNA (cffDNA) from maternal plasma for trisomies 21, 18, and 13 in twin pregnancies. Ninety-two women with twin pregnancies were recruited. The results were identified through karyotypes of amniocentesis or clinical examination and follow-up of the neonates. Fluorescent in-situ hybridization was used to examine the placentas postnatally in cases of false-positive results. The fetuses with autosomal trisomy 21 (n = 2) and trisomy 15 (n = 1) were successfully detected via MPS testing of cffDNA. There was one false-positive for trisomy 13 (n = 1), and fluorescence in-situ hybridization (FISH) identified confined placental mosaicism in this case. For twin pregnancies undergoing second-trimester screening for trisomy, MPS testing of cffDNA is feasible and can enhance the diagnostic spectrum of non-invasive prenatal testing, which could effectively reduce invasive prenatal diagnostic methods. In addition to screening for trisomy 21, 18, and 13 by cffDNA, MPS can detect fetal additional autosomal trisomy. False-positive results cannot completely exclude confined placental mosaicism.

Investigating Pregnancy Outcomes After Abnormal Cell-Free Fetal DNA Test Results [25F

INTRODUCTION: Cell-free fetal DNA (cfDNA) testing has increased sensitivity and specificity compared to other prenatal screening methods, but invasive diagnostic testing (IDT) is recommended for confirmation. We examined factors associated with pregnancy continuation or termination after abnormal cfDNA results. METHODS: We conducted a retrospective chart review of 39 women with abnormal cfDNA results between March 2012 and September 2015 at an urban academic hospital. We analyzed data using descriptive statistics, Fisher exact, and Wilcoxon rank-sum tests. RESULTS: Mean age was 33.9±7.6 years; 64% (25/39) were advanced maternal age and 67% (26/39) had abnormal ultrasounds. Median gestational age at time of cfDNA testing was 18 3/7 weeks (interquartile range 12 2/7, 20 5/7). CfDNA results included trisomy 21 (90%, 35/39), trisomy 18 (8%, 3/39), and both trisomy 21/monosomy X (3%, 1/39). Of 39 women, 22 (56%) continued and 10 (26%) terminated the pregnancy; six (15%) had fetal demises and one was lost to follow-up. For women continuing or terminating the pregnancy, pregnancy outcomes were not associated with cfDNA results (P&gt;.99), maternal age (P=.74), race (P=.23), or having abnormal ultrasounds (P=.68). Only 13 women underwent IDT; all had trisomy 21. Of women continuing their pregnancies, 55% (12/22) declined further genetic counseling and 73% (16/22) declined IDT. Most women terminating their pregnancies (80%, 8/10) underwent IDT. Not pursuing IDT was associated with continuing the pregnancy (P=.006). CONCLUSION: Over half of women with abnormal cfDNA results continued the pregnancy and declined IDT. Most women terminating their pregnancy pursued IDT, suggesting that women considering termination undergo more confirmatory tests.

Sex chromosome aneuploidy screening in a general population.

Cell-free fetal DNA testing is being used in parallel or in contingency screening as part of the first trimester screen. The test has high sensitivity and specificity for the trisomies 21, 18 and 13. The test also offers the option of assessing sex chromosome aneuploidies (SCA) which are recognised to be the next most common group of aneuploidies in the live birth population. Companies that offer the sex chromosome assessment report an accuracy rate of above 99% and a significant number of high-risk results have been detected in a multi-site Australian ultrasound practice. A high proportion of these women underwent prenatal testing to further assess the sex chromosomes. This study reports the results of these invasive investigations and results show that many of the high-risk SCA results appear to be false positives. This study reports the clinical experience of cell-free fetal DNA (cfDNA) testing with regard to sex chromosome aneuploidies in singleton pregnancies for a multi-site Sydney specialist O&G Ultrasound practice.

Cell-Free DNA Analysis of Targeted Genomic Regions in Maternal Plasma for Non-Invasive Prenatal Testing of Trisomy 21, Trisomy 18, Trisomy 13, and Fetal Sex.

There is great need for the development of highly accurate cost effective technologies that could facilitate the widespread adoption of noninvasive prenatal testing (NIPT). We developed an assay based on the targeted analysis of cell-free DNA for the detection of fetal aneuploidies of chromosomes 21, 18, and 13. This method enabled the capture and analysis of selected genomic regions of interest. An advanced fetal fraction estimation and aneuploidy determination algorithm was also developed. This assay allowed for accurate counting and assessment of chromosomal regions of interest. The analytical performance of the assay was evaluated in a blind study of 631 samples derived from pregnancies of at least 10 weeks of gestation that had also undergone invasive testing. Our blind study exhibited 100% diagnostic sensitivity and specificity and correctly classified 52/52 (95% CI, 93.2%-100%) cases of trisomy 21, 16/16 (95% CI, 79.4%-100%) cases of trisomy 18, 5/5 (95% CI, 47.8%-100%) cases of trisomy 13, and 538/538 (95% CI, 99.3%-100%) normal cases. The test also correctly identified fetal sex in all cases (95% CI, 99.4%-100%). One sample failed prespecified assay quality control criteria, and 19 samples were nonreportable because of low fetal fraction. The extent to which free fetal DNA testing can be applied as a universal screening tool for trisomy 21, 18, and 13 depends mainly on assay accuracy and cost. Cell-free DNA analysis of targeted genomic regions in maternal plasma enables accurate and cost-effective noninvasive fetal aneuploidy detection, which is critical for widespread adoption of NIPT.

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

Achondroplasia is generally detected by abnormal prenatal ultrasound findings in the third trimester of pregnancy and then confirmed by molecular genetic testing of fetal genomic DNA obtained by aspiration of amniotic fluid. This invasive procedure presents a small but significant risk for both the fetus and mother. Therefore, non-invasive procedures using cell-free fetal DNA in maternal plasma have been developed for the detection of the fetal achondroplasia mutations. To determine whether the fetus carries the de novo mis-sense genetic mutation at nucleotide 1138 in FGFR3 gene involved in >99% of achondroplasia cases, we developed two independent methods: digital-droplet PCR combined with minisequencing, which are very sensitive methods allowing detection of rare alleles. We collected 26 plasmatic samples from women carrying fetus at risk of achondroplasia and diagnosed to date a total of five affected fetuses in maternal blood. The sensitivity and specificity of our test are respectively 100% [95% confidence interval, 56.6-100%] and 100% [95% confidence interval, 84.5-100%]. This novel, original strategy for non-invasive prenatal diagnosis of achondroplasia is suitable for implementation in routine clinical testing and allows considering extending the applications of these technologies in non-invasive prenatal diagnosis of many other monogenic diseases. © 2016 John Wiley & Sons, Ltd.

Stakeholder attitudes and needs regarding cell-free fetal DNA testing.

To explore stakeholder views on cell-free DNA testing and highlight findings important for successful implementation and the provision of best practice in counseling. Noninvasive tests based on the analysis of cell-free fetal DNA are now widely available in clinical practice and applications are expanding rapidly. It is essential that stakeholder views are considered in order to identify and address any ethical and social issues. We provide an overview of stakeholder viewpoints and then focus on the key issues of informed decision making, test uptake, service delivery and information sources. Stakeholders are positive about the introduction of cell-free fetal DNA testing into clinical practice. They describe both practical and psychological benefits arising from tests that are safe and can potentially be performed earlier in pregnancy. Key concerns, which include the potential for these tests to have a negative impact on informed decision making and increased societal pressure to have testing, can be addressed through careful parent-directed counseling. As applications for these tests expand it is increasingly important to develop innovative approaches to facilitate good understanding for parents who are offered noninvasive prenatal testing.