Fetal Genotype Research Articles

Effects of fetal and maternal genotype on placentome morphology in sheep

Both maternal and fetal genotypes contribute to conceptus development. The objective was to determine how placentome number, size, and type and fetal weight was influenced after reciprocal embryo transfer in Columbia and Romanov sheep. Reciprocal embryo transfer was conducted between Columbia and Romanov ewes where a single embryo was transferred into Romanov and Columbia recipients [Romanov embryo in a Romanov uterus (RinR, n = 9); Romanov embryo in a Columbia uterus (RinC, n = 7); Columbia embryo in a Columbia uterus (CinC, n = 8); Columbia embryo in a Romanov uterus (CinR, n = 4)]. On day 130 of gestation, fetuses were weighed and placentomes were morphologically typed, weighed, and measured.Regardless of maternal genotype, Romanov fetuses were smaller (P < 0.05) compared to Columbia fetuses. Moreover, CinC fetuses were larger (P < 0.05) than CinR fetuses. There was a tendency (P = 0.12) for a fetal by maternal genotype interaction on total placentome weight, but main effects were significant for fetal genotype (P = 0.04) and maternal genotype (P < 0.01). The number of Type A placentomes was greater than any other types. Type A placentomes had a greater (P < 0.05) contribution to total placentome weight within the Romanov uterus, or when associated with a Romanov fetus, than within the Columbia breed, in which placentome type was evenly distributed. The hypothesis that the Romanov uterus would limit the growth of a Columbia conceptus is accepted; however, the Romanov conceptus did not experience augmented growth when transferred into a Columbia uterus as predicted.

Non-invasive fetal RHD genotyping

Immunization against RhD is the major cause of hemolytic disease of the fetus and newborn (HDFN), which causes fetal or neonatal death. The introduction of postnatal immune prophylaxis in the 1960s drastically reduced immunization incidents in pregnant, D-negative women. In several countries, antenatal prophylaxis is combined with postnatal prophylaxis to further minimize the immunization risk. Due to lack of knowledge of the fetal RhD type, antenatal prophylaxis is given to all D-negative women. In the European population, approximately 40% of pregnant women carry a D-negative fetus and are thus at no risk of immunization. Noninvasive fetal RhD genotyping enables antenatal prophylaxis to be targeted to only those women carrying a D-positive fetus to avoid unnecessary treatment. Based on an analysis of cell-free fetal DNA from the plasma of pregnant women, this approach has recently undergone technical improvements and rapid clinical implementation. As a screening assay, the sensitivity is >99.3% from a gestational age of approximately 10–11 weeks. In addition, fetal RhD genotyping is widely used to assess the risk of HDFN in anti-D immunized women.

Non-invasive fetal RHD genotyping: validation of the test and implementation of a screening program to guide anti-D prophylaxis

Non-invasive fetal RHD genotyping: validation of the test and implementation of a screening program to guide anti-D prophylaxis

Two Reliable Methodical Approaches for Non-Invasive RHD Genotyping of a Fetus from Maternal Plasma.

Noninvasive fetal RHD genotyping is an important tool for predicting RhD incompatibility between a pregnant woman and a fetus. This study aimed to assess a methodological approach other than the commonly used one for noninvasive fetal RHD genotyping on a representative set of RhD-negative pregnant women. The methodology must be accurate, reliable, and broadly available for implementation into routine clinical practice. A total of 337 RhD-negative pregnant women from the Czech Republic region were tested in this study. The fetal RHD genotype was assessed using two methods: real-time PCR and endpoint quantitative fluorescent (QF) PCR. We used exon-7-specific primers from the RHD gene, along with internal controls. Plasma samples were analyzed and measured in four/two parallel reactions to determine the accuracy of the RHD genotyping. The RHD genotype was verified using DNA analysis from a newborn buccal swab. Both methods showed an excellent ability to predict the RHD genotype. Real-time PCR achieved its greatest accuracy of 98.6% (97.1% sensitivity and 100% specificity (95% CI)) if all four PCRs were positive/negative. The QF PCR method also achieved its greatest accuracy of 99.4% (100% sensitivity and 98.6% specificity (95% CI)) if all the measurements were positive/negative. Both real-time PCR and QF PCR were reliable methods for precisely assessing the fetal RHD allele from the plasma of RhD-negative pregnant women.

Accurate fetal variant calling in the presence of maternal cell contamination.

High-throughput sequencing of fetal DNA is a promising and increasingly common method for the discovery of all (or all coding) genetic variants in the fetus, either as part of prenatal screening or diagnosis, or for genetic diagnosis of spontaneous abortions. In many cases, the fetal DNA (from chorionic villi, amniotic fluid, or abortive tissue) can be contaminated with maternal cells, resulting in the mixture of fetal and maternal DNA. This maternal cell contamination (MCC) undermines the assumption, made by traditional variant callers, that each allele in a heterozygous site is covered, on average, by 50% of the reads, and therefore can lead to erroneous genotype calls. We present a panel of methods for reducing the genotyping error in the presence of MCC. All methods start with the output of GATK HaplotypeCaller on the sequencing data for the (contaminated) fetal sample and both of its parents, and additionally rely on information about the MCC fraction (which itself is readily estimated from the high-throughput sequencing data). The first of these methods uses a Bayesian probabilistic model to correct the fetal genotype calls produced by MCC-unaware HaplotypeCaller. The other two methods "learn" the genotype-correction model from examples. We use simulated contaminated fetal data to train and test the models. Using the test sets, we show that all three methods lead to substantially improved accuracy when compared with the original MCC-unaware HaplotypeCaller calls. We then apply the best-performing method to three chorionic villus samples from spontaneously terminated pregnancies.

Using droplet digital PCR to screen for rare blood donors: Proof of principle

BackgroundDigital droplet PCR (ddPCR) is a very sensitive high throughput genotyping methodology. To date, the use of ddPCR in immunohematology is restricted to fetal genotyping of red blood cell antigens. Our hypothesis is that this technology could be applied to screen for rare red blood cell genotypes, such as Di(b-). MethodsNucleic acid of 3168 donors was extracted for viral screening routine in pools of 6, which were converted into three types of 48-donor pools: control pools (only DI*B/*B samples), pools with varying amount of DI*A/*B samples (n = 1–5) and a pool with one rare DI*A/*A sample. Pools were genotyped using ddPCR to detect and quantify DI*A and DI*B alleles. ResultsDI*A allele was accurately detected in all pools containing Di(a + b+) samples and in the pool containing one Di(a + b-) sample. No copies were detected in the control pools (n = 60). The ratio between the number of DI*A and DI*B copies varied significantly between the pools and the triplicates. ConclusionThe proposed ddPCR assay was accurate in identifying the rare DI*A allele in large pools of donors and can be applied to screen for Di(b-) phenotype. The strategy can potentially be extended to search for other rare RBC phenotypes.

A medical innovation: a new and improved method of DNA extraction with electrowetting-on-dielectric of genetic testing in-vitro fertilization (IVF)

The current technique for the detection of fetus genotype by in-vitro fertilization (IVF) is quite expensive and is unsuitable/ inconvenient for all patients and even affects the embryonic growth cycle. We thus propose a new method to extract small amount of cell-free DNA (cf-DNA) from embryo culture medium. We introduce a new format for DNA extraction depending on digital microfluidics (DMF), electrowetting-on-dielectric (EWOD) and magnetic forces to separate, suspend DNA-coated magnetic particles. Nano-droplets (of nano-liter size) are electrostatically controlled on an array of insulated electrodes by EWOD system. By applying voltage to the various electrodes, multiple nano-droplets can be manipulated precisely in the same device. This feature enables one to extract DNA at ultra-small concentrations (fg/μL to pg/μL) from an embryo culture medium with an EWOD system. We applied our EWOD platform to extract a small amount of cf-DNA from a clinical embryo culture medium of mouse. In the future, we will distinguish the DNA in the real embryo buffer according to the length of the telomere. This feature would be valuable for the selection of an embryo before an implantation when the gene information can be recognized.

Noninvasive Fetal Genotyping by Droplet Digital PCR to Identify Maternally Inherited Monogenic Diabetes Variants.

BackgroundBabies of women with heterozygous pathogenic glucokinase (GCK) variants causing mild fasting hyperglycemia are at risk of macrosomia if they do not inherit the variant. Conversely, babies who inherit a pathogenic hepatocyte nuclear factor 4a (HNF4A) diabetes variant are at increased risk of high birth weight. Noninvasive fetal genotyping for maternal pathogenic variants would inform pregnancy management.MethodsDroplet digital PCR was used to quantify reference and variant alleles in cell-free DNA extracted from blood from 38 pregnant women heterozygous for a GCK or HNF4A variant and to determine fetal fraction by measurement of informative maternal and paternal variants. Droplet numbers positive for the reference/ alternate allele together with the fetal fraction were used in a Bayesian analysis to derive probability for the fetal genotype. The babies’ genotypes were ascertained postnatally by Sanger sequencing.ResultsDroplet digital PCR assays for GCKor HNF4A variants were validated for testing in all 38 pregnancies. Fetal fraction of ≥2% was demonstrated in at least 1 cell-free DNA sample from 33 pregnancies. A threshold of ≥0.95 for calling homozygous reference genotypes and ≤0.05 for heterozygous fetal genotypes allowed correct genotype calls for all 33 pregnancies with no falsepositive results. In 30 of 33 pregnancies, a result was obtained from a single blood sample.ConclusionsThis assay can be used to identify pregnancies at risk of macrosomia due to maternal monogenic diabetes variants.

Implementation of high-throughput non-invasive prenatal testing for fetal RHD genotype testing in England: Results of a cross-sectional survey of maternity units and expert interviews.

BackgroundPreviously, routine antenatal anti‐D prophylaxis (RAADP) was administered to all RhD‐negative mothers to reduce the risk of sensitisation in the UK's National Health Service (NHS). If the baby is RhD‐negative, RAADP is not required. In 2016, the UK National Institute for Health and Care Excellence (NICE) recommended non‐invasive prenatal testing (NIPT) for fetal RHD genotype as a cost‐effective option to guide RAADP.ObjectivesTo evaluate the implementation of high‐throughput NIPT for fetal RHD genotype in maternity units in England by addressing research recommendations from the NICE. These were to reduce uncertainty around the resource use and cost of staff training, management of samples and results and record‐keeping, as well as resultant changes to antenatal or post‐partum care and performance of NIPT.MethodsA cross‐sectional survey was developed and sent to clinicians at 39 English NHS Trusts in May 2018. Qualitative interviews with seven individuals were conducted to explore missing or contraindicatory data. Qualitative findings were supplemented with NIPT test results (April 2017 to February 2019) from English hospitals.ResultsStaff reported that training took up to 30 minutes. There were no extra costs associated with sample management or additional appointments. Extra time required for record‐keeping and management of test results was balanced later in the patient pathway. The antenatal pathway was not changed in the Trusts surveyed. The survey revealed that four post‐partum scenarios were being used within English NHS Trusts. The frequency of inconclusive NIPT results was 4.3%.ConclusionNIPT for fetal RHD genotype can be implemented without consuming substantial extra resources through incorporation into an existing patient pathway.

Noninvasive prenatal diagnosis for Duchenne muscular dystrophy based on the direct haplotype phasing.

We aimed to investigate the validity of noninvasive prenatal diagnosis (NIPD) based on direct haplotype phasing without the proband or other family members and its feasibility for clinical application in the case of Duchenne muscular dystrophy (DMD). Thirteen singleton-pregnancy families affected by DMD were recruited. The pathogenic variants in the pregnant females have been identified by multiplex ligation-dependent probe amplification (MLPA). We resolved maternal haplotypes for each family by performing targeted linked-read sequencing of their high molecular weight DNA, respectively. Then, we integrated the maternal haplotypes and the targeted sequencing results of maternal plasma DNA to infer the fetal haplotype and the DMD gene variant status. The fetal genotypes were further validated by using chorionic villus sampling. The method of directly resolving maternal haplotype through targeted linked-read sequencing was smoothly performed in 12 participated families, but one failed (F11). The predicted variant status of 12 fetuses was correct, which had been confirmed by invasive prenatal diagnosis. Direct haplotyping of NIPD based on linked-read sequencing for DMD is accurate.

Non-invasive risk-assessment and bleeding prophylaxis with IVIG in pregnant women with a history of fetal and neonatal alloimmune thrombocytopenia: management to minimize adverse events

IntroductionIn pregnant women with a history of fetal and neonatal alloimmune thrombocytopenia (FNAIT), prenatal intervention in subsequent pregnancies may be required to prevent fetal bleeding. Several invasive and non-invasive protocols have been published: amniocentesis for fetal genotyping, fetal blood sampling for the determination of fetal platelet count, intrauterine platelet transfusions, and weekly maternal i.v. immunoglobulin (IVIG) infusion with or without additional corticosteroid therapy. This is the first retrospective study that report the experience with a non-invasive protocol focused on side effects of maternal IVIG treatment and neonatal outcome.MethodsPregnant women with proven FNAIT in history and an antigen positive fetus were treated with IVIG (1 g/kg/bw) every week. To identify potential IVIG-related hemolytic reactions isoagglutinin titer of each IVIG lot and maternal blood count were controlled. IVIG-related side effects were prospectively documented and evaluated. Furthermore, ultrasound examination of the fetus was performed before starting IVIG administration and continued regularly during treatment. Outcome of the index and subsequent pregnancy was compared. Corresponding data of the newborns were analyzed simultaneously.ResultsIVIG was started at 20 weeks of gestation (median). Compared to the index pregnancy, platelet counts of the newborns were higher in all cases. No intracranial hemorrhage occurred (Index pregnancies: 1 case). Platelet counts were 187 × 109/l (median, range 22–239, 95% CI) and one newborn had mild bleeding. No severe hemolytic reaction was observed and side effects were moderate.ConclusionAmong pregnant women with FNAIT history, the use of non-invasive fetal risk determination and maternal IVIG resulted in favorite outcome of all newborns. Invasive diagnostic or therapeutic procedures in women with a history of FNAIT should be abandoned.

Noninvasive prenatal diagnosis for pregnancies at risk for β-thalassaemia: a retrospective study.

To evaluate the clinical feasibility of noninvasive prenatal diagnosis (NIPD) for β-thalassaemia using circulating single molecule amplification and re-sequencing technology (cSMART). Through carrier screening, 102 pregnant Chinese couples carrying pathogenic HBB gene variants were recruited to the study. Pregnancies were managed using traditional invasive prenatal diagnosis (IPD). Retrospectively, we evaluated the archived pregnancy plasma DNA by NIPD to evaluate the performance of our cSMART assay for fetal genotyping. Chinese prenatal diagnostic centres specialising in thalassaemia testing. Chinese carrier couples at high genetic risk for β-thalassaemia. Fetal cell sampling was performed by amniocentesis and HBB genotypes were determined by reverse dot blot. NIPD was performed by a newly designed HBB cSMART assay and fetal genotypes were called by measuring the allelic ratios in the maternal cell-free DNA. Concordance of HBB fetal genotyping between IPD and NIPD and the sensitivity and specificity of NIPD. Invasive prenatal diagnosis identified 29 affected homozygotes or compound heterozygotes, 54 heterozygotes and 19 normal homozygotes. Compared with IPD results, 99 of 102 fetuses (97%) were correctly genotyped by our NIPD assay. Two of three discordant samples were false positives and the other sample involved an incorrect call of a heterozygote carrier as a homozygote normal. Overall, the sensitivity and specificity of our NIPD assay was 100% (95% CI 88.06-100.00%) and 97.26% (95% CI 90.45-99.67%), respectively. This study demonstrates that our cSMART-based NIPD assay for β-thalassaemia has potential clinical utility as an alternative to IPD for pregnant HBB carrier couples. A new noninvasive test for pregnancies at risk for β-thalassaemia.

Impact of cell-free fetal DNA on early invasive prenatal diagnosis at a Chinese reference maternal medicine center

Purpose The aim of this study is to evaluate the impact of the utility of maternal cell-free DNA (cfDNA) on the number of chorionic villus sampling (CVS) at a mainland Chinese maternal hospital. Methods This was a retrospective cohort study conducted in consecutive singleton pregnancies that underwent CVS between the 11th and 14th gestational weeks at a Chinese maternal hospital during a nine-year period. The indications, complications and prenatal diagnosis results were evaluated. Results This study consisted of 5108 CVS procedures, including 2000 performed for fetal karyotyping, and 3108 performed for fetal single-gene genotyping. During the period with the introduction of cfDNA, the proportion of the number of CVS procedures for the indication of positive serum screening declined significantly, and abnormal ultrasound was the main indication for CVS performed for fetal karyotyping. Thalassemia was always the main indication for CVS, accounting for 50.5% of all CVS cases. Conclusions cfDNA has changed the spectrum of CVS indications. CVS is now the invasive procedure performed for patients with a fetus having a very high risk of fetal genetic defects, including fetuses having major abnormal ultrasound or having a risk of single-gene disorder inherited from their parents.

Association of preeclampsia with infant APOL1 genotype in African Americans

BackgroundBlack women in the United States and Africa are at an increased risk for preeclampsia. Allelic variants in the gene for apolipoprotein LI, APOL1, are found only in populations of African ancestry, and have been shown to contribute significant risk for kidney disease. Recent studies suggest these APOL1 variants also may contribute risk for preeclampsia.MethodsThe association of preeclampsia with carriage of APOL1 risk alleles was evaluated in a case-control study of deliveries from black women at a single center in Cleveland, Ohio that included gross and histopathologic evaluations of placental tissues (395 cases and 282 controls). Using logistic regression models, associations between fetal APOL1 genotype and preeclampsia were evaluated using several case definitions based on prematurity and severity of preeclampsia, with uncomplicated term pregnancies as controls. Associations between APOL1 genotype and pathological features were also examined.ResultsThe infant APOL1 genotype was significantly associated with preeclampsia in a dominant inheritance pattern with odds ratio of 1.41 (P=0.029, 95% CI 1.037, 1.926). Stratifying preeclampsia cases by preterm birth, significant associations were detected for both recessive (O.R.=1.70, P=0.038) and additive (O.R.=1.33, P=0.028) inheritance patterns. APOL1 genotype, however, was not significantly associated with pathological changes or other perinatal observations.ConclusionsPreeclampsia appears to be another disease associated with APOL1 variants, however, further studies are needed to increase confidence in the mode of inheritance. By understanding the association of APOL1 variants with preeclampsia, genetic screening tests for APOL1 may be useful to predict at-risk pregnancies and targeted interventions may be developed to improve pregnancy outcomes.

Molecular diagnosis in recessive pediatric neurogenetic disease can help reduce disease recurrence in families

BackgroundThe causes for thousands of individually rare recessive diseases have been discovered since the adoption of next generation sequencing (NGS). Following the molecular diagnosis in older children in a family, parents could use this information to opt for fetal genotyping in subsequent pregnancies, which could inform decisions about elective termination of pregnancy. The use of NGS diagnostic sequencing in families has not been demonstrated to yield benefit in subsequent pregnancies to reduce recurrence. Here we evaluated whether genetic diagnosis in older children in families supports reduction in recurrence of recessive neurogenetic disease.MethodsRetrospective study involving families with a child with a recessive pediatric brain disease (rPBD) that underwent NGS-based molecular diagnosis. Prenatal molecular testing was offered to couples in which a molecular diagnosis was made, to help couples seeking to prevent recurrence. With this information, families made decisions about elective termination. Pregnancies that were carried to term were assessed for the health of child and mother, and compared with historic recurrence risk of recessive disease.ResultsBetween 2010 and 2016, 1172 families presented with a child a likely rPBD, 526 families received a molecular diagnosis, 91 families returned to the clinic with 101 subsequent pregnancies, and 84 opted for fetal genotyping. Sixty tested negative for recurrence for the biallelic mutation in the fetus, and all, except for one spontaneous abortion, carried to term, and were unaffected at follow-up. Of 24 that genotyped positive for the biallelic mutation, 16 were electively terminated, and 8 were carried to term and showed features of disease similar to that of the older affected sibling(s). Among the 101 pregnancies, disease recurrence in living offspring deviated from the expected 25% to the observed 12% ([95% CI 0·04 to 0·20], p = 0·011).ConclusionsMolecular diagnosis in an older child, coupled with prenatal fetal genotyping in subsequent pregnancies and genetic counselling, allows families to make informed decisions to reduce recessive neurogenetic disease recurrence.

Prevalence of Partial Hydatidiform Mole in Products of Conception From Gestations With Fetal Triploidy Merits Reflex Genotype Testing Independent of the Morphologic Appearance of the Chorionic Villi.

Diagnosis of first-trimester partial mole is challenging as the key morphologic features may not be well-developed and may overlap with those of a nonmolar gestation harboring a cytogenetic disorder or degenerative changes. Genotype testing has emerged as the reference tool to distinguish partial mole (diandric triploid genotype) from its nonmolar mimics. However, observer variation in defining the minimum threshold of how much morphologic alteration is required to trigger genotype testing may result in a subset of partial moles that go undetected. We hypothesized that the results of fetal aneuploidy testing performed for prenatal screening or evaluation of miscarriage may assist with triggering molecular testing in the evaluation of products of conception, specifically if fetal triploidy is detected. Gestations with fetal triploidy are either a partial mole (diandric triploidy) or are nonmolar (digynic triploidy). The aims of this study were to define the prevalence of partial mole in 20 products of conception specimens with known fetal triploidy by performing genotype testing and then to determine how well established morphologic criteria for partial mole correlate with the genotype results in this setting. Genotype testing demonstrated that 65% (13/20) were a partial mole and the remainder were nonmolar digynic triploid gestations. Most partial moles were under 9 weeks gestational age and, as expected, lacked classic well-developed morphologic features. Nearly a third (4/13) of the partial moles were originally interpreted as normal or nonmolar gestations with minimal abnormalities that did not merit molecular testing to exclude a partial mole. Even with the retrospective systematic morphologic review, only 23% (3/13) exhibited the combination of chorionic villous enlargement of ≥2.5 mm and cisterns, which has been previously established as the morphologic criteria with the highest predictive value for a molecularly defined partial mole. The other 77% exhibited focal, limited, variable degrees and extent of villous morphologic alterations. We conclude that, given the high prevalence of partial mole among products of conception with known fetal triploidy and the low prevalence of diagnostic morphologic findings in such specimens, reflex genotype testing should be performed in all such cases, regardless of whether or not the morphologic features are suspicious for a partial mole. This reflex testing strategy mitigates against the subjectivity of determining whether subtle villous abnormalities are significant enough to merit pursuing genotype testing. The success of this strategy depends on the clinician documenting the fetal triploidy result at the time of submitting the products of conception specimen and therefore clinician education is needed. Finally, it remains to be determined whether the risk for postmolar gestational trophoblastic disease is the same in diandric triploid gestations that exhibit classic morphologic features as in those that exhibit minimal or negligible villous morphologic abnormalities.

External quality assessment of noninvasive fetal RHD genotyping.

Fetal RHD genotyping of cell-free maternal plasma DNA from RhD negative pregnant women can be used to guide targeted antenatal and postnatal anti-D prophylaxis for the prevention of RhD immunization. To assure the quality of clinical testing, we conducted an external quality assessment workshop with the participation of 31 laboratories. Aliquots of pooled maternal plasma from gestational week 25 were sent to each laboratory. One sample was fetal RHD positive, and a second sample was fetal RHD negative. A reporting scheme was supplied for data collection, including questions regarding the methodological setup, results and clinical recommendations. The samples were tested blindly. Different methodological approaches were used; 29 laboratories used qPCR and two laboratories used ddPCR, employing a total of eight different combinations of RHD exon targets. Fetal RHD genotyping was performed with no false-negative and no false-positive results. One inconclusive result was reported for the RHD positive sample. All clinical conclusions were satisfactory. This external quality assessment workshop demonstrates that despite the different approaches taken to perform the clinical assays, fetal RHD genotyping is a reliable laboratory assay to guide targeted use of Rh prophylaxis in a clinical setting.

Noninvasive prenatal paternity testing by means of SNP-based targeted sequencing.

ObjectiveTo develop a method for noninvasive prenatal paternity testing based on targeted sequencing of single nucleotide polymorphisms (SNPs).MethodSNPs were selected based on population genetics data. Target‐SNPs in cell‐free DNA extracted from maternal blood (maternal cfDNA) were analyzed by targeted sequencing wherein target enrichment was based on multiplex amplification using QIAseq Targeted DNA Panels with Unique Molecular Identifiers. Fetal SNP genotypes were called using a novel bioinformatics algorithm, and the combined paternity indices (CPIs) and resultant paternity probabilities were calculated.ResultsFetal SNP genotypes obtained from targeted sequencing of maternal cfDNA were 100% concordant with those from amniotic fluid‐derived fetal genomic DNA. From an initial panel of 356 target‐SNPs, an average of 148 were included in paternity calculations in 15 family trio cases, generating paternity probabilities of greater than 99.9999%. All paternity results were confirmed by short‐tandem‐repeat analysis. The high specificity of the methodology was validated by successful paternity discrimination between biological fathers and their siblings and by large separations between the CPIs calculated for the biological fathers and those for 60 unrelated men.ConclusionThe novel method is highly effective, with substantial improvements over similar approaches in terms of reduced number of target‐SNPs, increased accuracy, and reduced costs.

Nondestructive Identification of Rare Trophoblastic Cells by Endoplasmic Reticulum Staining for Noninvasive Prenatal Testing of Monogenic Diseases.

Noninvasive prenatal detection of monogenic diseases based on cell‐free DNA is hampered by challenges in obtaining a sufficient fraction and adequate quality of fetal DNA. Analyzing rare trophoblastic cells from Papanicolaou smears carrying the entire fetal genome provides an alternative method for noninvasive detection of monogenic diseases. However, intracellular labeling for identification of target cells can affect the quality of DNA in varying degrees. Here, a new approach is developed for nondestructive identification of rare fetal cells from abundant maternal cells based on endoplasmic reticulum staining and linear discriminant analysis (ER‐LDA). Compared with traditional methods, ER‐LDA has little effect on cell quality, allowing trophoblastic cells to be analyzed on the single‐cell level. Using ER‐LDA, high‐purity of trophoblastic cells can be identified and isolated at single cell resolution from 60 pregnancies between 4 and 38 weeks of gestation. Pathogenic variants, including –SEA/ deletion mutation and point mutations, in 11 fetuses at risk for α‐ or β‐thalassemia can be accurately detected by this test. The detection platform can also be extended to analyze the mutational profiles of other monogenic diseases. This simple, low‐cost, and noninvasive test can provide valuable fetal cells for fetal genotyping and holds promise for prenatal detection of monogenic diseases.

Liquid biopsy for management of haemolytic disease of the fetus and newborn

Background: Haemolytic disease of the fetus and newborn arises from maternal allo-antibodies to red cell antigens, crossing the placenta. The most frequent antibodies after RhD, are Kell, Duffy and RhCE antigens. Liquid biopsy in pregnancy management is concerned with the detection of cell-free fetal DNA (cffDNA) circulating in the maternal plasma. The accuracy for fetal RHD genotyping is well established. Aim: To evaluate a maternal blood test to predict the fetal, non-RhD, blood group status. Methods: A clinical collaborative study assessed the accuracy for a suite of assays to predict fetal Kell, Duffy, Rhc/C and Rhe/E status for women with respective antibodies (n=90, gestation age 8–37 weeks). Droplet digital PCR technology, ddPCR, tested for single nucleotide variants (SNVs) associated with these blood group antigens. Infant cord blood serotypes were used as the primary outcome measure. Results: Fetal specific signals were detected as early as 8 weeks gestation. Genotyping matched all available infant cord outcomes 45/45. Accuracy is 100% (95% confidence interval 91.96–100%). Discussion: A clinical demand exists for liquid biopsy of maternal blood for fetal blood group genotyping. Droplet digital PCR is accurate and provides an evidence base to guide clinical care for at-risk pregnancies.