Non-invasive Prenatal Diagnostic Testing Research Articles

Enhancing fetal outcomes in GCK-MODY pregnancies: a precision medicine approach via non-invasive prenatal GCK mutation detection.

Mutations in the GCK gene cause Maturity Onset Diabetes of the Young (GCK-MODY) by impairing glucose-sensing in pancreatic beta cells. During pregnancy, managing this type of diabetes varies based on fetal genotype. Fetuses carrying a GCK mutation can derive benefit from moderate maternal hyperglycemia, stimulating insulin secretion in fetal islets, whereas this may cause macrosomia in wild-type fetuses. Modulating maternal glycemia can thus be viewed as a form of personalized prenatal therapy, highly beneficial but not justifying the risk of invasive testing. We therefore developed a monogenic non-invasive prenatal diagnostic (NIPD-M) test to reliably detect the transmission of a known maternal GCK mutation to the fetus. A small amount of fetal circulating cell-free DNA is present in maternal plasma but cannot be distinguished from maternal cell-free DNA. Determining transmission of a maternal mutation to the fetus thus implies sequencing adjacent polymorphisms to determine the balance of maternal haplotypes, the transmitted haplotype being over-represented in maternal plasma. Here we present a series of such tests in which fetal genotype was successfully determined and show that it can be used to guide therapeutic decisions during pregnancy and improve the outcome for the offspring. We discuss several potential hurdles inherent to the technique, and strategies to overcome these. Our NIPD-M test allows reliable determination of the presence of a maternal GCK mutation in the fetus, thereby allowing personalized in utero therapy by modulating maternal glycemia, without incurring the risk of miscarriage inherent to invasive testing.

Cell-Free Fetal DNA and Non-Invasive Prenatal Diagnosis of Chromosomopathies and Pediatric Monogenic Diseases: A Critical Appraisal and Medicolegal Remarks.

Cell-free fetal DNA (cffDNA) analysis is a non-invasive prenatal diagnostic test with a fundamental role for the screening of chromosomic or monogenic pathologies of the fetus. Its administration is performed by fetal DNA detection in the mother's blood from the fourth week of gestation. Given the great interest regarding its validation as a diagnostic tool, the authors have set out to undertake a critical appraisal based on a wide-ranging narrative review of 45 total studies centered around such techniques. Both chromosomopathies and monogenic diseases were taken into account and systematically discussed and elucidated. Not surprisingly, cell-free fetal DNA analysis for screening purposes is already rather well-established. At the same time, considerable interest in its diagnostic value has emerged from this literature review, which recommends the elaboration of appropriate validation studies, as well as a broad discourse, involving all stakeholders, to address the legal and ethical complexities that such techniques entail.

Isolation and Enrichment of Circulating Fetal Cells for NIPD: An Overview.

Prenatal diagnosis plays a crucial role in clinical genetics. Non-invasive prenatal diagnosis using fetal cells circulating in maternal peripheral blood has become the goal of prenatal diagnosis, to obtain complete fetal genetic information and avoid risks to mother and fetus. The development of high-efficiency separation technologies is necessary to obtain the scarce fetal cells from the maternal circulation. Over the years, multiple approaches have been applied, including choice of the ideal cell targets, different cell recovering technologies, and refined cell isolation yield procedures. In order to provide a useful tool and to give insights about limitations and advantages of the technologies available today, we review the genetic research on the creation and validation of non-invasive prenatal diagnostic testing protocols based on the rare and labile circulating fetal cells during pregnancy.

Precision medicine in diabetes: A non-invasive prenatal diagnostic test for the determination of fetal glucokinase mutations.

ABSTRACTHyperglycemia caused by mutations in the glucokinase gene, GCK, is the most common form of monogenic diabetes. Prenatal diagnosis is important, as it impacts on treatment. This study reports a monogenic non‐invasive prenatal diagnostic (NIPD‐M) test on cell‐free DNA in maternal plasma using the relative haplotype dosage. In three pregnancies of two families with known maternal GCK mutations, the fetal genotype was determined unambiguously already at 12 weeks of gestation. In summary, proof is provided of the feasibility for NIPD‐M in GCK diabetes.

Is cervical swab an efficient method for developing a new noninvasive prenatal diagnostic test for numerical and structural chromosome anomalies?

Background/aim Prenatal diagnosis is vital to obtain healthy generation for risky pregnancies. There have been several approaches, some of which are routinely applied in clinics to evaluate the possible prenatal deficiencies and/or diseases. In the present study, we aimed to isolate the fetal cells from endocervical samples and try to identify possible anomalies which were proved by Amniocentesis (AS) and chorionic villus sampling (CVS) methods.Materials and methodsEndoservical specimens were collected from 100 pregnant women. Cells were separated in parallel by fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) using human leukocyte antigen (HLA) G233 and placental alkaline phosphatase (PLAP) antibodies. CMA (comprehensive meta-analysis) were carried out and male fetuses were confirmed with Sex determining region Y (SRY) amplification.Results The percent of HLA G233 and placental and placental alkaline phosphatase (PLAP) positive cells were 4.55% and 84.59%, respectively. The percent of cells positive for both markers was 14.75%. CMA analyses were not informative. (SRY) was amplified in 67% of the samples.Conclusion However, the success rate of the both cell sorting and scanning of DNA anomalies by aCGH and/or RT-PCR was limited, preventing the applicability of this proposal in the clinics. Still, the success of the proposed method depends on the development of the novel fetal cell-specific antibodies and the improvements in the sorting systems.

OP16.10: Prenatal diagnosis of aberrant right subclavian artery (ARSA) in unselected population: our own experience

The aim of our study was to analyses all cases with aberrant right subclavian artery (ARSA) and its association with chromosomal and other congenital malformations. 2863 fetuses between 12 and 34 weeks were examined by ultrasound in our centre between January 2015 and March 2017 by one examiner. ARSA was assessed in the axial plane on the level of three vessels and trachea view. ARSA was identified in 33 fetuses. ARSA was an isolated finding in 13 (39%) fetuses and non-isolated in 20 (60%) fetuses. In 9 (27%) of these 33 cases ARSA associated with congenital cardiac anomalies such as AVSD, coarctation of aorta, vascular ring, right aortic arch, HLHS, bilateral superior vena cava and tetralogy of Fallot (2). Additional ultrasound findings such as an occipital encefalocele, hemivertebra (2 cases), diastematomyelia, partial agenesis of corpus callosum, cavum velli interposity, tricuspid regurgitation, single umbilical artery, large NT (3 cases), abnormal hands, retrograde ductus venosus blood flow were present in 14 (42%) fetuses. 5 (15%) of these of 33 were aneuploidies: Trisomy 21 was diagnosed in 4 fetuses, Trisomy 18 in 1 fetus. 22q11 microdeletion was detected in 1 fetus. All women with isolated ARSA were recommended to have a non-invasive prenatal diagnostic test. No cases with Down syndrome were detected in all 13 fetuses with isolated ARSA. Isolated ARSA does not seem to be associated with a significantly increased risk of aneuploidy, however using of a non-invasive diagnostic test might be discussed. When ARSA is identified a detailed anatomy scan should be recommended. An invasive procedure with genetic diagnosis should be discussed when additional ultrasound abnormalities or risk factors for aneuploidy are found.

186: Non-invasive prenatal testing (NIPT) versus diagnostic testing for evaluation of fetal structural anomalies

186: Non-invasive prenatal testing (NIPT) versus diagnostic testing for evaluation of fetal structural anomalies

Separating sheep from goats: a European view on the patent eligibility of biomedical diagnostic methods

The Supreme Court’s decisions in Mayo, Myriad and Alice, as well as the CAFC’s in Roslin focused widespread attention on the formulation of patent-eligibility exclusions for specific biological material and diagnostic methods. The debate recently intensified with the CAFC’s Sequenom decision and denial of a rehearing en banc. The claims at issue in U.S. Patent No. 6,258,540 (“US ’540 patent”) are directed to methods of genetic testing by detecting and amplifying paternally inherited fetal cell-free DNA (cffDNA) from maternal blood and plasma. Before the development of this non-invasive prenatal diagnostic test, patients were placed at higher risk and maternal plasma was routinely discarded as waste. A reluctant CAFC formulaically interpreted the Supreme Court-devised bifurcated test to identify patent ineligible subject matter and invalidated the patent for this ground-breaking method. Notably, Judge Linn wrote that this innovation deserves patent protection, but that the “sweeping language of the test” established in Mayo requires a determination that the claims are patent ineligible. On March 21, 2016 Sequenom Inc. filed for certiorari and the issue may once again find itself at the Supreme Court. As framed by Sequenom, the question presented is: Whether a novel method is patent-eligible where: (1) a researcher is the first to discover a natural phenomenon; (2) that unique knowledge motivates him to apply a new combination of known techniques to that discovery; and (3) he thereby achieves a previously impossible result without preempting other uses of the discovery? Interestingly, in Europe the EPO upheld essentially the same claims. European equivalents of the patents considered in Myriad, Mayo, Alice and Roslin were also treated differently than in the US. Hence, these cases undermine the global integration of patent standards and provide fodder for discussing patentability requirements at an international level. Referring to these developments, our paper discusses these issues from a comparative European perspective. Section 1 provides a very brief summary of the European patent framework and case law regarding medical diagnostic methods. Leaving aside national peculiarities that would exceed the limitations of this study we focus on the EPO’s patent eligibility approach vis-a-vis medical diagnostic methods similar to those in Sequenom v. Ariosa. Section 2 discusses our findings and the differences between the US and European approaches from a broader innovation and patent policy perspective providing the basis for concluding remarks in section 3.

An easy test but a hard decision: ethical issues concerning non-invasive prenatal testing for autosomal recessive disorders.

Prenatal testing based on cell-free fetal DNA in maternal serum is now possible for specific monogenic conditions, and studies have shown that the use of non-invasive testing is supported by prospective parents and health professionals. However, some ethical issues have been raised concerning informed consent and paternal rights. The objective of this study was to explore ethical aspects of the use of non-invasive prenatal diagnostic testing for autosomal recessive disorders. We used a qualitative cross-sectional design, based on Thematic Analysis, and recruited 27 individuals of reproductive age who were carriers of one of four conditions: thalassaemia, sickle cell disease, cystic fibrosis or spinal muscular atrophy. Data were collected via focus groups or interviews. Participants were aware of the potential for such tests to be viewed as routine and suggested that obtaining written consent and allowing time for consideration is needed to facilitate autonomous choice and informed consent. All participants felt that mothers should be able to request such tests, but fathers who declined carrier testing should be made aware that fetal test results may reveal their status. We suggest that a written record of consent for non-invasive prenatal diagnosis should be used as a standard to help reinforce the serious nature of the test results. Where the father's carrier status could be revealed through fetal testing, he should be made aware of this before the results are available. Health professionals should discuss with the pregnant woman the best way to manage unsought information about the father's carrier status to minimise family disruption.

Complexity of noninvasive prenatal screening and diagnostic testing for an unbalanced translocation involving chromosomes 5 and 18

What's already known about this topic? Noninvasive prenatal screening (NIPS) via cell-free DNA analysis of maternal blood has been validated for detection of Trisomy 18 with a sensitivity of 97.4% (95% CI: 93–100%) and false positive rate of 0.07% (95% CI: 0.0–0.3%). (Confidence intervals provided are for Ariosa Diagnostics as this lab was involved with our case; however, the sensitively and specificity of NIPS vary slightly depending on the company performing the testing). Parental balanced reciprocal translocations are known to result in the potential for offspring with unbalanced chromosome material and significant effects on physical and intellectual development, as well as lifespan. What does this study add? A case report of NIPS results when a fetus has an unbalanced translocation involving one of the chromosomes that is being tested via cell-free DNA. Natural history information of a child born with Cri-du-Chat syndrome and partial Trisomy 18 concurrently. © 2013 John Wiley & Sons, Ltd.

Pränataldiagnostik

During the last few years, there has been a rapid development in prenatal diagnosis. Due to the improvements in sonographic examinations and the introduction of first-trimester screening, the number of invasive prenatal diagnostic procedures has dropped by more than 50 %. Recently, noninvasive prenatal diagnostic tests with cell-free fetal DNA from maternal blood have also become available and will further enhance this development. As invasive prenatal procedures will become less frequent in the near future, the proportion of procedure-related abortions will further decrease.

Genetic Testing: New Challenges in the Healthcare of Women

Dr Wera Hofmann is an expert in biochemistry and has over 12 years of human genetic diagnostics experience. Until 2006, she supervised a diagnostic unit for BRCA gene testing at the Interdisciplinary Center for Hereditary Breast Cancer (Max Delbrück Center, Berlin, Germany). She has also been the Managing Director of the Professional Association of German Human Geneticists, BVDH, which is a trade association. In 2008, Hofmann became a Medical Director at LifeCodexx (Konstanz, Germany), where she has worked on the development of a noninvasive prenatal diagnostic test that detects chromosomal aneuploidies in fetuses.

Akonni TruTip® and Qiagen® Methods for Extraction of Fetal Circulating DNA - Evaluation by Real-Time and Digital PCR

Due to the low percentage of fetal DNA present in maternal plasma (< 10%) during early gestation, efficient extraction processes are required for successful downstream detection applications in non-invasive prenatal diagnostic testing. In this study, two extraction methods using similar chemistries but different workflows were compared for isolation efficiency and percent fetal DNA recovery. The Akonni Biosystems TruTip technology uses a binding matrix embedded in a pipette tip; the Circulating Nucleic Acids Kit from Qiagen employs a spin column approach. The TruTip method adds an extra step to decrease the recovery of DNA fragments larger than 600 bp from the sample to yield an overall higher percentage of smaller molecular weight DNA, effectively enriching for fetal DNA. In this evaluation, three separate extraction comparison studies were performed - a dilution series of fragmented DNA in plasma, a set of clinical maternal samples, and a blood collection tube time point study of maternal samples. Both extraction methods were found to efficiently extract small fragment DNA from large volumes of plasma. In the amended samples, the TruTip extraction method was ~15% less efficient with overall DNA recovery, but yielded an 87% increase in % fetal DNA relative to the Qiagen method. The average percent increase of fetal DNA of TruTip extracted samples compared to the Qiagen method was 55% for all sets of blinded clinical samples. A study comparing extraction efficiencies from whole blood samples incubated up to 48 hours prior to processing into plasma resulted in more consistent % fetal DNA recoveries using TruTip. The extracted products were tested on two detection platforms, quantitative real-time PCR and droplet digital PCR, and yielded similar results for both extraction methods.

Clinical applications of maternal plasma fetal DNA analysis: translating the fruits of 15 years of research

The collection of fetal genetic materials is required for the prenatal diagnosis of fetal genetic diseases. The conventional methods for sampling fetal genetic materials, such as amniocentesis and chorionic villus sampling, are invasive in nature and are associated with a risk of fetal miscarriage. For decades, scientists had been pursuing studies with goals to develop non-invasive methods for prenatal diagnosis. In 1997, the existence of fetal derived cell-free DNA molecules in plasma of pregnant women was first demonstrated. This finding provided a new source of fetal genetic material that could be obtained safely through the collection of a maternal blood sample and provided a new avenue for the development of non-invasive prenatal diagnostic tests. Now 15 years later, the diagnostic potential of circulating fetal DNA analysis has been realized. Fruitful research efforts have resulted in the clinical implementation of a number of non-invasive prenatal tests based on maternal plasma DNA analysis and included tests for fetal sex assessment, fetal rhesus D blood group genotyping and fetal chromosomal aneuploidy detection. Most recently, research groups have succeeded in decoding the entire fetal genome from maternal plasma DNA analysis which paved the way for the achievement of non-invasive prenatal diagnosis of many single gene diseases. A paradigm shift in the practice of prenatal diagnosis has begun.

Non-invasive prenatal diagnostic test accuracy for fetal sex using cell-free DNA a review and meta-analysis

BackgroundCell-free fetal DNA (cffDNA) can be detected in maternal blood during pregnancy, opening the possibility of early non-invasive prenatal diagnosis for a variety of genetic conditions. Since 1997, many studies have examined the accuracy of prenatal fetal sex determination using cffDNA, particularly for pregnancies at risk of an X-linked condition. Here we report a review and meta-analysis of the published literature to evaluate the use of cffDNA for prenatal determination (diagnosis) of fetal sex. We applied a sensitive search of multiple bibliographic databases including PubMed (MEDLINE), EMBASE, the Cochrane library and Web of Science.ResultsNinety studies, incorporating 9,965 pregnancies and 10,587 fetal sex results met our inclusion criteria. Overall mean sensitivity was 96.6% (95% credible interval 95.2% to 97.7%) and mean specificity was 98.9% (95% CI = 98.1% to 99.4%). These results vary very little with trimester or week of testing, indicating that the performance of the test is reliably high.ConclusionsBased on this review and meta-analysis we conclude that fetal sex can be determined with a high level of accuracy by analyzing cffDNA. Using cffDNA in prenatal diagnosis to replace or complement existing invasive methods can remove or reduce the risk of miscarriage. Future work should concentrate on the economic and ethical considerations of implementing an early non-invasive test for fetal sex.

Non-Invasive Prenatal Genetic Testing: A Study of Public Attitudes

Background/Objectives: Non-invasive prenatal genetic diagnostic (NIPD) tests are being developed using cell-free fetal DNA in the maternal circulation. NIPD tests avoid or reduce the need for invasive diagnostic procedures for conditions like Down syndrome. Discussion of ethical and social implications of these techniques is increasing. We report findings from a study of public attitudes relevant to the introduction of NIPD. A key aim was to examine the range of attitudes relevant to NIPD within a diverse sample. Methods: Qualitative analysis of written free text ‘first responses’ to a written neutral description of NIPD as part of a Q-methodology study conducted with a purposive sample of the UK population (n = 71). Results: The majority (63%) of respondents described their first response as positive. However, respondents displayed ambivalence, expressing positive views of individual/medical rationale for NIPD and unease concerning public health rationale and societal implications. Unease related to eugenic reasoning underlying existing prenatal testing, ‘too much control’ in reproduction, commercial provision, information and support requirements for expanded testing, and limiting the use of testing. Conclusions: These findings suggest that regulating and monitoring commercial provision of NIPD services, and monitoring introduction and clinical use, are a public preference.

Detection of chromosomal aneuploidies in fetal cells isolated from maternal blood using single‐chromosome dual‐probe FISH analysis

Detection of chromosomal aneuploidies using fetal cells isolated from maternal blood, for prenatal non-invasive genetic investigation, has been a long-sought goal of clinical genetics to replace amniocentesis and chorionic villous sampling to avoid any risk to the fetus. The purpose of this study was to develop a sensitive and specific new assay for diagnosing aneuploidy with circulating fetal cells isolated from maternal blood as previously reported using two novel approaches: (i) simultaneous immunocytochemistry (ICC) evaluation using a monoclonal antibody for i-antigen, followed by fluorescence in situ hybridization (FISH); (ii) dual-probe FISH analysis of interphase nuclei using two differently labeled probes, specific for different loci of chromosomes 21 and 18; in addition, short tandem repeats (STR) analysis on single cells isolated by micromanipulation was applied to confirm the presence of fetal cells in the cell sample enriched from maternal blood. Blood samples were obtained from women carrying trisomic fetuses, and from non-pregnant women and men as controls. Using ICC-FISH approach, a large heterogeneity in immunostaining pattern was observed, which is a source of very subjective signal interpretation. Differently, dual-probe FISH analysis provided for a correct diagnosis of all pregnancies: the mean percentage of trisomic cells was 0.5% (range, 0.36-0.76%), while the mean percentage of trisomic cells in the control group (normal pregnancies or non-pregnant women) was ≤0.20%. The application of the dual-probe FISH protocol on fetal cells isolated from maternal blood enables accurate molecular detection of fetal aneuploidy, thus providing a foundation for development of non-invasive prenatal diagnostic testing.

Multiplexed Analysis of Circulating Cell-Free Fetal Nucleic Acids for Noninvasive Prenatal Diagnostic RHD Testing

Objective The objective of the study was the evaluation of a novel multiplex assay to detect fetal Rh blood group D-antigen gene ( RHD) loci in maternal plasma from RhD-negative, pregnant women. Study Design An RHD genotyping assay was designed to detect exons 4, 5, 7, and 10 and RHDΨ (pseudogene) of the RHD gene along with a Y chromosome–specific assay and a generic polymerase chain reaction amplification control. Plasma samples from 150 RhD-negative pregnant women were assayed for fetal RHD genotype using the MassARRAY system. Results The fetal RHD status of 148 of 150 samples (98.7%) was correctly classified; 86 (57.3%) and 62 (41.3%) were positive and negative, respectively. Conclusion This study demonstrates that noninvasive prenatal diagnostics with a single-reaction multiplexed assay is a viable path toward routine characterization of fetal RHD genotypes using circulating cell-free fetal DNA in maternal plasma on the MassARRAY system and is perhaps preferable to serologic testing as currently used clinically.

Multiplexed analysis of circulating cell-free fetal nucleic acids for noninvasive prenatal diagnostic RHD testing

The objective of the study was the evaluation of a novel multiplex assay to detect fetal Rh blood group D-antigen gene (RHD) loci in maternal plasma from RhD-negative, pregnant women. An RHD genotyping assay was designed to detect exons 4, 5, 7, and 10 and RHDΨ (pseudogene) of the RHD gene along with a Y chromosome-specific assay and a generic polymerase chain reaction amplification control. Plasma samples from 150 RhD-negative pregnant women were assayed for fetal RHD genotype using the MassARRAY system. The fetal RHD status of 148 of 150 samples (98.7%) was correctly classified; 86 (57.3%) and 62 (41.3%) were positive and negative, respectively. This study demonstrates that noninvasive prenatal diagnostics with a single-reaction multiplexed assay is a viable path toward routine characterization of fetal RHD genotypes using circulating cell-free fetal DNA in maternal plasma on the MassARRAY system and is perhaps preferable to serologic testing as currently used clinically.

Non-invasive prenatal diagnosis by fetal nucleic acid analysis in maternal plasma: the coming of age

Prenatal diagnosis is an important part of obstetrics care. In the current prenatal programmes, definitive diagnosis of fetal genetic or chromosomal conditions is conducted through fetal sampling by amniocentesis or chorionic villus sampling. To obviate the risks of fetal miscarriage that are associated with the invasive sampling procedures, we have been developing non-invasive prenatal diagnostic tests based on cell-free fetal DNA analysis from maternal plasma. To date, fetal sex and rhesus D status determination by circulating fetal DNA analysis is performed clinically in many centres. Strategies for the non-invasive diagnosis of monogenic diseases have been developed. Accurate detection of fetal trisomy 21 by next-generation sequencing has been achieved. Many of the non-invasive prenatal tests could be introduced to the clinics as soon as cost-effective and high throughput protocols are developed.