DNA In Maternal Plasma Research Articles

Multiple z-Score Based Method for Noninvasive Prenatal Test Using Cell-Free DNA in Maternal Plasma

Objective: To improve the detecting accuracy of chromosomal aneuploidy of fetus by non-invasive prenatal testing (NIPT) using next generation sequencing data of pregnant women’s cell-free DNA. Methods: We proposed the multi-Z method which uses 21 z-scores for each autosomal chromosome to detect aneuploidy of the chromosome, while the conventional NIPT method uses only one z-score. To do this, mapped read numbers of a certain chromosome were normalized by those of the other 21 chromosomes. Average and standard deviation (SD), which are used for calculating z-score of each sample, were obtained with normalized values between all autosomal chromosomes of control samples. In this way, multiple z-scores can be calculated for 21 autosomal chromosomes except oneself. Results: Multi-Z method showed 100% sensitivity and specificity for 187 samples sequenced to 3 M reads while the conventional NIPT method showed 95.1% specificity. Similarly, for 216 samples sequenced to 1 M reads, Multi-Z method showed 100% sensitivity and 95.6% specificity and the conventional NIPT method showed a result of 75.1% specificity. Conclusion: Multi-Z method showed higher accuracy and robust results than the conventional method even at low coverage reads.

Non-invasive prenatal diagnosis (NIPD) of cystic fibrosis: an optimized protocol using MEMO fluorescent PCR to detect the p.Phe508del mutation

BackgroundAnalysis of cell-free foetal DNA (cff-DNA) in maternal plasma is very promising for early diagnosis of monogenic diseases; in particular, cystic fibrosis (CF). However, NIPD of single-gene disorders has been limited by the availability of suitable technical platforms and the need to set up patient or disease-specific custom-made approaches. MethodsTo make research applications more readily accessible to the clinic, we offer a simple assay combining two independent methods to determine the presence or absence of paternally inherited foetal allele p.Phe508del (the most frequent mutation in CF patients worldwide). The first method detects the presence or absence of a p.Phe508del allele by Mutant Enrichment with 3′-Modified Oligonucleotide PCR coupled to Fragment Length Analysis (MEMO-PCR-FLA). The second method detects the p.Phe508del allele with classical Multiplex Fluorescent PCR including five intragenic and extragenic STR markers of the CFTR locus and a specific SRY sequence. ResultsWe collected 24 plasma samples from 23 women carrying foetuses at risk for CF and tested each sample using both methods. Our new procedures were successfully applied to 10 couples where fathers carried the p.Phe508del mutation and mothers were carrying a different mutation in the CFTR gene. These simple tests provided clear positive or negative results from the maternal plasma of the pregnant women. We confirmed the presence of cff-DNA in the studied samples by the identification of a tri-allelic DNA profile using a miniSTR kit. All results were correlated with chorionic villus sampling or amniocentesis analyses. ConclusionsThis NIPD approach, easily set up in any clinical laboratory where prenatal diagnosis is routinely performed, offers many advantages over current methods: it is simple, rapid, and cost-effective. It opens up the possibility for testing a large number of couples with offspring at risk for CF.

Cost‐effectiveness of implementing molecular immunohematology

BackgroundMolecular immunohematology is practised in clinical laboratories and blood centres. It is based on red cell genotyping, which is a technology whereby DNA‐based techniques are used to evaluate genes for the particular single and multiple nucleotide substitutions, deletions, insertions and gene conversions that determine the expression of red cell antigens. Fetal red cell genotyping, and in particular RHD genotyping, using the discarded cell pellet of amniotic fluid was developed some 20 years ago. It replaced the need for fetal blood sampling and also can determine paternal zygosity with better certainty. Cell‐free fetal DNA in maternal plasma is now the non‐invasive (fetal) alternative to amniotic fluid as the biological material of choice. It is being used in Europe to reduce the exposure of Rh immune globulin for the routine Rh‐negative pregnancy. RHD genotyping is used to determine zygosity and resolve weak D antigen discrepancies as a cost equivalent alternative to the administration of Rh immune globulin. Since then, a plethora of data has accumulated on the value of screening donors for rare blood types. For common antigens, red cell genotyping as an historical test‐of‐record will create efficiencies in the labelling of antigen‐negative blood if care is taken in testing appropriate repeat donors. Certainly, it has been shown that exposing antigen‐negative types through the entire supply chain replaces shipping from a central facility.ConclusionMolecular immunohematology is not the implementation of cost reductions but the recognition of ‘effective’ processes and algorithms that incorporate molecular testing to provide tangible benefits to pregnant women and transfusion recipients.

Genomic Analysis of Plasma DNA in Prenatal Medicine and Beyond

The discovery of cell-free fetal DNA in maternal plasma has enabled the development of noninvasive prenatal testing (NIPT). With the increased use of NIPT globally, there have been a number of reports of plasma DNA aberrations that have orignated from maternal malignancies, rather than signifying genomic aberrations in the fetus. In this regard, it would be highly desirable to develop a technology for tracing the tissue of origin of genomic aberrations observed in plasma. Hence, we have developed a technology called plasma DNA tissue mapping which allows this to be achieved. Through the development of over 5800 methylation markers, we are able to deduce the tissue of origin of plasma DNA molecules. We have demonstrated the utility of this approach by tracing genomic aberrations observed in the plasma of a pregnant woman during the course of NIPT to a B-cell lymphoma. We believe that this technology would be broad applications in medical research and diagnostics. DisclosuresLo:Cirina Limited: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Xcelom Limited: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Illumina: Patents & Royalties; Sequenom: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

Towards the Development of a Noninvasive Prenatal Test for Beta-Thalassemia: Utilization of Probe Capture Enrichment and Next Generation Sequencing

β-thalassemia causes significant morbidity and mortality worldwide. Currently, the diagnosis can be made prenatally for couples at risk using invasive procedures such as chorionic villus sampling and amniocentesis. Noninvasive prenatal testing (NIPT) on maternal blood samples would enable earlier fetal diagnosis and eliminate risks associated with these procedures.The discovery of cell-free fetal DNA (cfFDNA) in maternal plasma and advances in next generation sequencing (NGS) have made NIPT a clinical reality for aneuploidies. Diagnosing autosomal recessive (AR) disorders is more challenging as it requires determination of both the paternally and maternally inherited alleles and can be particularly difficult when both parents carry the same mutation. Previous studies used methods to identify the paternally inherited allele in maternal plasma through the detection of a DNA sequence variant present in the father and absent in the mother. Our method expands the target region beyond the β-globin gene into highly polymorphic regions to increase the likelihood of identifying unique paternal sequences. Unlike the detection of the paternal allele, there is no direct qualitative approach for determining the maternally inherited allele. Our solution is an indirect quantitative method that compares the ratio of allelic sequence reads to infer which maternal allele was inherited by the fetus.We have developed a novel NGS assay which utilizes probe capture enrichment, a method employing thousands of short overlapping oligonucleotide probes complementary to a target sequence region. This design makes it uniquely applicable to short, fragmented DNA, such as cell-free DNA (~150 base pairs). Our probe assay targets a contiguous 900 bp region which spans a portion of the β-globin gene (part of exon 2 as well as the entirety of IVS-1 and exon 1) and extends 579 bp into the highly polymorphic 5' UTR region to identify linked sequence variations. Additionally, the assay targets 451 single nucleotide polymorphisms (SNPs) throughout the genome. SNPs that are "informative" (i.e. an allele present in the fetus and absent in the mother) are used to calculate the fraction of cfDNA from the fetus.We have evaluated our assay’s performance in a set of experiments designed to simulate the challenging aspects of cfFDNA: very low quantity and short fragment size. The assay’s sensitivity was tested by preparing libraries containing amounts of DNA ranging from 50 to 0.05 ngs. At DNA amounts as low as 5 ng (5-fold lower than that expected in plasma), 100% coverage was achieved for the targeted β-globin region and SNPs. The probe/NGS system successfully captured and sequenced DNA fragments as short as 35 bps and as little as 0.5 ng of DNA with >95% coverage. To test the ability of the probe/NGS system to resolve mixtures, DNA samples from patients with known β-globin mutations were combined in ratios ranging from 2.5:97.5 to 20:80 in 25 ng total DNA to mimic the maternal/fetal cfDNA mixture. Our assay was able to detect minority heterozygous mutant alleles at proportions as low as 1.25% and 0.3 ng of DNA. In a mixture designed to simulate a case with a shared parental mutation (CD41/42(-TTCT)), we identified a linked SNP (rs713040) allele, which was within 250 bp of the mutation and unique to the minor fraction. We used this SNP to distinguish the CD41/42 (-TTCT) mutation contributed by the minor fraction.Based on 6 mixtures, we observed 24.3% (110/451) of SNPs to be informative, allowing for a precise estimate of the minor fraction. We estimated the minor fraction to be 11.88% and 5.98% compared to the expected 10% and 5%, respectively. To show proof of concept for inferring the minor ("fetal") genotype when the major ("maternal") genotype was heterozygous mutant (IVS1-5 (G>C)), the estimated minor fraction (10.4% based on 104 informative SNPs) was used to calculate the expected allelic ratios of the 3 different possible minor ("fetal") genotypes. The minor genotype was correctly inferred as wt/wt based on the observed mixture ratio (42.56/57.41 mut/wt) compared to the expected (44.93/55.07).These data show that our probe capture/NGS system can overcome the challenges implicit in the analysis of cfFDNA for NIPT: low DNA amount (<5 ng) and short fragments (<150 bp). We expect that our approach using the fetal fraction estimate will allow us to successfully infer the fetal genotype when applied to maternal plasma. DisclosuresErlich:Allen and Overy, Law Office: Consultancy.

Second generation noninvasive fetal genome analysis reveals de novo mutations, single-base parental inheritance, and preferred DNA ends

Plasma DNA obtained from a pregnant woman was sequenced to a depth of 270× haploid genome coverage. Comparing the maternal plasma DNA sequencing data with the parental genomic DNA data and using a series of bioinformatics filters, fetal de novo mutations were detected at a sensitivity of 85% and a positive predictive value of 74%. These results represent a 169-fold improvement in the positive predictive value over previous attempts. Improvements in the interpretation of the sequence information of every base position in the genome allowed us to interrogate the maternal inheritance of the fetus for 618,271 of 656,676 (94.2%) heterozygous SNPs within the maternal genome. The fetal genotype at each of these sites was deduced individually, unlike previously, where the inheritance was determined for a collection of sites within a haplotype. These results represent a 90-fold enhancement in the resolution in determining the fetus's maternal inheritance. Selected genomic locations were more likely to be found at the ends of plasma DNA molecules. We found that a subset of such preferred ends exhibited selectivity for fetal- or maternal-derived DNA in maternal plasma. The ratio of the number of maternal plasma DNA molecules with fetal preferred ends to those with maternal preferred ends showed a correlation with the fetal DNA fraction. Finally, this second generation approach for noninvasive fetal whole-genome analysis was validated in a pregnancy diagnosed with cardiofaciocutaneous syndrome with maternal plasma DNA sequenced to 195× coverage. The causative de novo BRAF mutation was successfully detected through the maternal plasma DNA analysis.

Ethical and Social Implications of Current Prenatal Genetic Testing

Abstract: A new prenatal genetic testing technique using cell-free fetal DNA in maternal plasma, the Non-Invasive Prenatal Test (or NIPT), was introduced in Japan in 2013. The NIPT is easy and safe but not definitive; it also has high sensitivity and specificity under certain conditions. In the near future, it could be used not only for detecting chromosomal aneuploidy, but also complete genome analysis of the fetus. However, termination of pregnancy is the only option for those wanting to avoid having a baby with a chromosomal anomaly or a genetic disease. Given this situation, this review explores the ethical and social issues surrounding prenatal genetic testing focusing on three points: 1) selective abortion as an ethical issue, 2) informed consent and decision making, and 3) alternative perspectives on prenatal testing.

An Advanced Model to Precisely Estimate the Cell-Free Fetal DNA Concentration in Maternal Plasma.

BackgroundWith the speedy development of sequencing technologies, noninvasive prenatal testing (NIPT) has been widely applied in clinical practice for testing for fetal aneuploidy. The cell-free fetal DNA (cffDNA) concentration in maternal plasma is the most critical parameter for this technology because it affects the accuracy of NIPT-based sequencing for fetal trisomies 21, 18 and 13. Several approaches have been developed to calculate the cffDNA fraction of the total cell-free DNA in the maternal plasma. However, most approaches depend on specific single nucleotide polymorphism (SNP) allele information or are restricted to male fetuses.MethodsIn this study, we present an innovative method to accurately deduce the concentration of the cffDNA fraction using only maternal plasma DNA. SNPs were classified into four maternal-fetal genotype combinations and three boundaries were added to capture effective SNP loci in which the mother was homozygous and the fetus was heterozygous. The median value of the concentration of the fetal DNA fraction was estimated using the effective SNPs. A depth-bias correction was performed using simulated data and corresponding regression equations for adjustments when the depth of the sequencing data was below 100-fold or the cffDNA fraction is less than 10%.ResultsUsing our approach, the median of the relative bias was 0.4% in 18 maternal plasma samples with a median sequencing depth of 125-fold. There was a significant association (r = 0.935) between our estimations and the estimations inferred from the Y chromosome. Furthermore, this approach could precisely estimate a cffDNA fraction as low as 3%, using only maternal plasma DNA at the targeted region with a sequencing depth of 65-fold. We also used PCR instead of parallel sequencing to calculate the cffDNA fraction. There was a significant association (r = 98.2%) between our estimations and those inferred from the Y chromosome.

Noninvasive Prenatal Paternity Testing (NIPAT) through Maternal Plasma DNA Sequencing: A Pilot Study.

Short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) have been already used to perform noninvasive prenatal paternity testing from maternal plasma DNA. The frequently used technologies were PCR followed by capillary electrophoresis and SNP typing array, respectively. Here, we developed a noninvasive prenatal paternity testing (NIPAT) based on SNP typing with maternal plasma DNA sequencing. We evaluated the influence factors (minor allele frequency (MAF), the number of total SNP, fetal fraction and effective sequencing depth) and designed three different selective SNP panels in order to verify the performance in clinical cases. Combining targeted deep sequencing of selective SNP and informative bioinformatics pipeline, we calculated the combined paternity index (CPI) of 17 cases to determine paternity. Sequencing-based NIPAT results fully agreed with invasive prenatal paternity test using STR multiplex system. Our study here proved that the maternal plasma DNA sequencing-based technology is feasible and accurate in determining paternity, which may provide an alternative in forensic application in the future.

Cell-free DNA in maternal plasma and serum: A comparison of quantity, quality and tissue origin using genomic and epigenomic approaches

ObjectivesThe objectives of this study were to compare the concentrations, size profiles and major tissue contributors of cell-free DNA (cfDNA) in plasma and in serum. Design and methodsThirteen pregnant women in the third trimester were recruited for this study. We collected EDTA-plasma and serum samples using various collection tubes. We determined their cfDNA concentrations and fetal cfDNA fractions using a zinc-finger X (ZFX)/zinc-finger Y (ZFY) droplet digital polymerase chain reaction (ZFX/ZFY ddPCR) assay. We used paired-end massively parallel sequencing (MPS) to measure plasma and serum cfDNA sizes at single-base resolution. We deconvoluted the genome-wide bisulfite sequencing data with reference to the methylation profiles of different tissues. ResultsThe concentrations of cfDNA collected in Sarstedt Serum Z tubes were found to be significantly higher than those in Greiner Bio-One Vacuette® Z Serum Separator Clot Activator tubes or Vacuette® Z Serum Clot Activator tubes. The concentrations of fetal cfDNA were significantly reduced in samples collected in the Vacuette® serum collection tubes. Fetal cfDNA fractions were significantly reduced in all sera compared to plasma. MPS of serum cfDNA revealed a right shift of the size distributions compared to plasma. Methylation-based tissue mapping of serum cfDNA revealed an increase of cfDNA from neutrophils and B cells but not T cells. ConclusionsThe use of different serum collection tubes has a significant impact on serum cfDNA concentrations. This effect is likely mediated through the combined effect of genomic DNA release from white blood cells and DNA degradation or removal.

Commentary on Down syndrome screening in China.

Prenatal screening for fetal Down syndrome has been widely practised in China for many years. In this special issue of BJOG, Tu et al. have published a systematic review on the performance of prenatal screening tests using biochemical and sonographic markers in Chinese women (Tu et al. BJOG 2016). Among the 78 included studies, of which most were from China, the majority (n = 64, 82%) were based on second-trimester biochemical markers. Although the pooled sensitivity and specificity for the second-trimester markers were 0.79 and 0.96, respectively, there were wide variations among individual studies. Most interestingly, 13 of the 64 second-trimester studies had a sensitivity of over 90%. Such an excellent performance has rarely been reported in studies from the non-Chinese population (Alldred et al. Cochrane Database Syst Rev 2012 Jun 13;(6):CD009925). This raised a concern over the quality of individual studies that warranted detailed analysis. In contrast, Li et al. reported in detail how to implement successfully a first-trimester combined screening (FTCS) programme for fetal Down syndrome in a resource-limited area of mainland China (Li et al. BJOG 2016). Despite the resource limitations, the author had followed strictly all the steps that were important for a successful screening programme, namely training and accreditation of staff, generation and use of local data, development of a customised cut-off and continuous quality assurance for both sonographers and biochemical assays. Specifically, the quality assurance steps included the following: (1) all sonographers were accredited by the Fetal Medicine Foundation and re-certified annually; (2) the performance of each sonographer was continuously monitored using a rolling standardised audit quality assurance report; (3) the multiple of the median values of individual biochemical markers were assessed monthly to determine the central tendency and dispersion; and (4) outcome data were followed-up. The above steps allowed early identification of deviation from expected performance for which early intervention could be initiated. Certification and quality assurance of sonographers is a particularly difficult process, requiring the support and cooperation of hospital administrators. It is interesting that in Li et al.'s study the cost of FTCS was quoted to be RMB800. Based on screening performance, noninvasive prenatal testing (NIPT) for fetal Down syndrome by cell-free DNA in maternal plasma is a far better test than FTCS, having a sensitivity and a specificity both above 99%. The cost of NIPT has come down substantially over the past 2 years. In China, NIPT is available in many major cities at a current user price most commonly between RMB2000 and 3000. It should not be long before the cost of NIPT is comparable to that of FTCS. At present, many hospitals in China are developing their own in-house NIPT platforms. This is a suboptimal move because such small hospital-based laboratories will not have enough volume to maintain a meaningful quality assurance programme. It is advisable that these screening programmes are coordinated within a region and centralised in one or a few centres where a strict quality assurance programme could be implemented. Even if NIPT has replaced conventional screening, the concept of ‘quality assurance’ as demonstrated in the study by Li et al. remains the key to the success for all screening programmes. None declared. Completed disclosure of interests form available to view online as supporting information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Debates on fetal fraction measurement and DNA-based noninvasive prenatal screening: time for standardisation?

Debates on fetal fraction measurement and DNA-based noninvasive prenatal screening: time for standardisation?

Cell-Free Placental DNA in Maternal Plasma in Relation to Placental Health and Function

Background: While cell-free placental DNA (cfp-DNA) increases in response to certain pathological conditions, confounding variables, such as placental size, may also contribute to its release. Furthermore, the relationship between cfp-DNA and maternal serum proteins has not been well investigated. Objective: To analyze plasma cfp-DNA levels and correlate with measurable placental parameters, maternal serum proteins, or pathologic conditions reflecting placental dysfunction. Method: Methylated fraction of RASSF1A was quantified in maternal plasma as a measure of cfp-DNA in a cohort of 86 pregnant women. Results: Placental dimensions or weight had no impact on cfp-DNA levels in noncomplicated pregnancies (n = 63). However, an association between β-hCG and cfp-DNA levels (p = 0.0012) was detected. Complications occurred in 23 pregnancies including chromosomal abnormalities, gestational hypertension, intrauterine growth restriction, and preterm birth. There was overall a skewed distribution (<-1 SD or >1 SD from mean) for cfp-DNA in the abnormal group, although due to the small number of samples for each pathology, we provide only descriptive data to assess possible trends in cfp-DNA variation. Conclusion: While cfp-DNA levels outside of the normal range may reflect placental distress, this relationship may be masked by a number of physiological confounders. The independence of cfp-DNA from β-hCG levels commonly assessed in pregnancy need to be further addressed.

Does Formaldehyde Increase Cell Free DNA in Maternal Plasma Specimens?

There have been conflicting observations reported in the literature regarding the effects of formaldehyde in the recovery of cell free fetal DNA (CFF DNA) from maternal plasma. The aim of the present study was to assess the effect of formaldehyde treatment on circulating cell free DNA. We conducted this study using blood specimens collected from 11 pregnant women, each of whom was carrying a male fetus. DYS14 and HBB real time assays were performed to quantify fetal and total circulating cell free DNA from formaldehyde treated and untreated maternal plasma specimens, respectively. The concentration of total circulating cell free DNA in formaldehyde-treated maternal plasma was reduced, compared with untreated maternal plasma (n = 11; P = .02). The percentage of CFF DNA between formaldehyde-treated and untreated maternal plasma specimens did not differ significantly (n = 11; P = .15). Addition of formaldehyde does not significantly enhance the proportion of cell free fetal DNA when blood specimens are processed without delay.

Effects of breed, age and season on quality of frozen-thawed semen in stallions

The aim of this investigation was to develop a real-time polymerase chain reaction (PCR) assay for detecting circulating cell-free fetal DNA in Arabian pregnant mare plasma and predicting fetal sex through identifying SRY gene. Peripheral blood samples of 28 pregnant mares were collected into tubes containing EDTA, at a single time point, between 8 and 20 weeks of gestation. Circulating cell-free fetal DNA was extracted from 3 mL of maternal plasma. Using outer and inner primers, real-time PCR was performed for the SRY gene and for the glyceraldehyde-3-phosphate dehydrogenase gene as the internal control. Amplicons were labeled as positive, negative, or inconclusive according to threshold cycle and melting curve analysis. Of the total 28 Arabian pregnant mares, 16 were carrying male and 12 were carrying female fetuses. On first round of real-time PCR, results were inconclusive for SRY gene; however, on the second round, conclusive results were obtained. In total, the accuracy of test was 88% (22/25) (confidence interval [CI]: 65.5, 95.6); it had a sensitivity of 85.7% (CI: 65.6, 92.5), whereas its specificity was 90.9% (CI: 65.3, 99.5). We had a positive predictive value of 92.3% (CI: 70.7, 99.6) and a negative predictive value of 83.3% (CI: 59.9, 91.2). To conclude, SRY gene can permit detection of equine fetal sex with a good accuracy by analyzing cell-free fetal DNA in maternal plasma just after 8 weeks of gestation.

Expanding non-invasive prenatal testing beyond chromosomes 21, 18, 13, X and Y.

Non-invasive prenatal testing (NIPT) based on cell-free DNA in maternal plasma is being expanded to include additional chromosome abnormalities beyond those involving chromosomes 21, 18, 13, X and Y. Review of population cytogenetic data provides insight into the likely number of additional abnormalities detectable. Additional clinically significant and cytogenetically recognizable abnormalities are present in less than 0.1% of newborns but clinically significant, or potentially significant, sub-microscopic imbalances are expected to be present in 1.7%. Cytogenetic studies on chorionic villus samples suggests that after excluding abnormalities involving chromosomes 21, 18, 13, X and Y, approximately 0.6% of NIPT results may be positive for an unbalanced abnormality attributable to mosaicism but most of these will not be confirmed at amniocentesis or in newborns. NIPT has also been developed for specific microdeletion syndromes and initial experience is now available. Laboratory procedures such as deeper sequencing and additional data analytics are rapidly evolving but even with existing protocols, it is already clear that NIPT does not necessarily need to be limited to trisomies 21, 18, 13 and the sex-chromosome abnormalities. Patient educational materials and genetic counseling services need to be available for women offered expanded NIPT.

Non-Invasive Prenatal Diagnosis of Lethal Skeletal Dysplasia by Targeted Capture Sequencing of Maternal Plasma.

BackgroundSince the discovery of cell-free foetal DNA in the plasma of pregnant women, many non-invasive prenatal testing assays have been developed. In the area of skeletal dysplasia diagnosis, some PCR-based non-invasive prenatal testing assays have been developed to facilitate the ultrasound diagnosis of skeletal dysplasias that are caused by de novo mutations. However, skeletal dysplasias are a group of heterogeneous genetic diseases, the PCR-based method is hard to detect multiple gene or loci simultaneously, and the diagnosis rate is highly dependent on the accuracy of the ultrasound diagnosis. In this study, we investigated the feasibility of using targeted capture sequencing to detect foetal de novo pathogenic mutations responsible for skeletal dysplasia.Methodology/Principal FindingsThree families whose foetuses were affected by skeletal dysplasia and two control families whose foetuses were affected by other single gene diseases were included in this study. Sixteen genes related to some common lethal skeletal dysplasias were selected for analysis, and probes were designed to capture the coding regions of these genes. Targeted capture sequencing was performed on the maternal plasma DNA, the maternal genomic DNA, and the paternal genomic DNA. The de novo pathogenic variants in the plasma DNA data were identified using a bioinformatical process developed for low frequency mutation detection and a strict variant interpretation strategy. The causal variants could be specifically identified in the plasma, and the results were identical to those obtained by sequencing amniotic fluid samples. Furthermore, a mean of 97% foetal specific alleles, which are alleles that are not shared by maternal genomic DNA and amniotic fluid DNA, were identified successfully in plasma samples.Conclusions/SignificanceOur study shows that capture sequencing of maternal plasma DNA can be used to non-invasive detection of de novo pathogenic variants. This method has the potential to be used to facilitate the prenatal diagnosis of skeletal dysplasia.

Cell-Free Total and Fetal DNA in First Trimester Maternal Serum and Subsequent Development of Preeclampsia

Objective The objective of this study was to assess the relationship between first trimester cell-free total and fetal DNA in maternal plasma and the subsequent development of preeclampsia. Study Design Nested case-control study of patients enrolled in the Combined Antioxidant and Preeclampsia Prediction Studies prediction study of 175 women who did and 175 women who did not develop preeclampsia. The predictive values of cell-free total and fetal DNA and the subsequent development of preeclampsia were measured using receiver operating characteristic curves. Results Cell-free total DNA was higher in African American (median; 25-75%; 6.15; 0.14-28.73; p = 0.02) and Hispanic (4.95; 0.20-26.82; p = 0.037) compared with white women (2.33; 0.03-13.10). Levels of cell-free total DNA were also associated with maternal body mass index (BMI) (p = 0.02). Cell-free total DNA levels were similar between women who later developed preeclampsia (3.52; 0.11-25.3) and controls (3.74; 0.12-21.14, p = 0.96). Conclusion There is no significant difference in levels of cell-free total DNA in the first trimester in women who subsequently develop preeclampsia. Levels of cell-free total DNA in the first trimester are increased in African American and Hispanic compared with white women, and levels increase with increasing BMI.

Diagnostic accuracy of fetal rhesus D genotyping using cell-free fetal DNA during the first trimester of pregnancy

Rhesus D genotyping with cell-free fetal DNA currently is used throughout the world. Although this technique has spread rapidly, its optimal use is still a matter of debate. This screening test has been introduced mainly for the treatment of RhD-negative pregnant women during the third trimester of pregnancy, thereby avoiding systematic anti-D prophylaxis, yet such a strategy has proved cost-ineffective. Publications reporting on fetal RHD genotyping with cell-free DNA in maternal plasma, specifically during the first trimester of pregnancy, are scarce in the scientific literature. This study sought to assess the performance of noninvasive fetal Rhesus D genotyping in the first trimester of pregnancy with a single-exon real-time polymerase chain reaction assay. This was a retrospective observational multicenter study. Cell-free fetal DNA was extracted from maternal blood of both nonimmunized and immunized women at 10-14 weeks of gestation. RHD sequence was determined by quantitative polymerase chain reaction, with amplification of exon 10. Results were compared with RhD phenotype data that were obtained by cord blood sampling of neonates. In total, 416 serum samples from RhD-negative pregnant women were collected during the first trimester of pregnancy. The test's overall sensitivity and specificity were 100% (95% confidence interval, 96.9-100.0) and 95.2% (95% confidence interval, 90.5-97.6), respectively. The negative and positive predictive values were 99.8% (95% confidence interval, 94.9-100.0) and 97.1% (95% confidence interval, 94.2-98.6), respectively. Fetal RHD status was inconclusive in 9 cases (2.2%). Noninvasive fetal RHD determination by single-exon quantitative polymerase chain reaction during the first trimester of pregnancy exhibits high accuracy.

Fetal-specific hypermethylated RASSF1A quantification in pregnancy

Objective: To quantify cell free fetal DNA (cffDNA) with fetal specific epigenetic marker, hypermethylated RASSF1A, in maternal plasma of normal pregnant women from 20 weeks of gestation and to assess its relationship with maternal age, height, pre-pregnancy weight and body mass index (BMI).Methods: Hundred normal pregnant women within the gestational age of 21–40 weeks were randomly selected and grouped into five (n = 20). Group 1: 21–24, Group 2: 25–28, Group 3: 29–32, Group 4: 33–36 and Group 5: 37–40 weeks. Maternal plasma DNA was extracted, digested with methylation-sensitive restriction enzyme, BstUI and the fetal specific DNA (cffDNA) was quantified by Real-time polymerase chain reaction (qRT-PCR).Results: The mean hypermethylated RASSF1A concentrations in different gestational groups were Group 1: 30.1 ± 14.9, Group 2: 52.6 ± 22.18, Group 3: 93.2 ± 19.08, Group 4: 172.8 ± 26.81 and Group 5: 337.8 ± 52.9 copies/ml. Pearson’s correlation analysis showed highly significant positive correlation between cffDNA and gestational age (r = 0.899, p < 0.001). BMI was also found to be positively related to cffDNA (r = 0.217, p < 0.05). However, it did not show any correlation with maternal age, height and pre-pregnancy weight.Conclusions: The gestational age-dependent increase of hypermethylated RASSF1A; the fetal specific epigenetic marker in maternal plasma was demonstrated, in an Indian study group of normal pregnant women. Findings would form the basis of future studies involving pregnancy complications that would aid in the early diagnosis of placental pathologies with hypermethylated RASSF1A.