Analysis Of DNA In Maternal Plasma Research Articles

Single-molecule sequencing reveals a large population of long cell-free DNA molecules in maternal plasma

In the field of circulating cell-free DNA, most of the studies have focused on short DNA molecules (e.g., <500 bp). The existence of long cell-free DNA molecules has been poorly explored. In this study, we demonstrated that single-molecule real-time sequencing allowed us to detect and analyze a substantial proportion of long DNA molecules from both fetal and maternal sources in maternal plasma. Such molecules were beyond the size detection limits of short-read sequencing technologies. The proportions of long cell-free DNA molecules in maternal plasma over 500 bp were 15.5%, 19.8%, and 32.3% for the first, second, and third trimesters, respectively. The longest fetal-derived plasma DNA molecule observed was 23,635 bp. Long plasma DNA molecules demonstrated predominance of A or G 5' fragment ends. Pregnancies with preeclampsia demonstrated a reduction in long maternal plasma DNA molecules, reduced frequencies for selected 5' 4-mer end motifs ending with G or A, and increased frequencies for selected motifs ending with T or C. Finally, we have developed an approach that employs the analysis of methylation patterns of the series of CpG sites on a long DNA molecule for determining its tissue origin. This approach achieved an area under the curve of 0.88 in differentiating between fetal and maternal plasma DNA molecules, enabling the determination of maternal inheritance and recombination events in the fetal genome. This work opens up potential clinical utilities of long cell-free DNA analysis in maternal plasma including noninvasive prenatal testing of monogenic diseases and detection/monitoring of pregnancy-associated disorders such as preeclampsia.

Antenatal screening for fetal trisomies using microarray-based cell-free DNA testing: A systematic review and meta-analysis.

To evaluate the test accuracy of non-invasive prenatal testing (NIPT) for fetal trisomy 21, 18, and 13 using cell-free (cf) DNA analysis in maternal plasma with microarray quantitation. Systematic review and meta-analysis. Searches in MEDLINE, Pre-MEDLINE, EMBASE, Web of Science, and the Cochrane Library to 09.07.2018. Five studies analyzing 3074 samples, including 187 trisomy 21, 43 trisomy 18, and 19 trisomy 13 cases, were identified. Risk of bias was high in all studies, introduced particularly by exclusions from analysis and by the role of the sponsor. Sensitivity of microarray-based cfDNA testing was 99.5% (95%CI 96.3%-99.9%) for trisomy 21, 97.7% (95%CI 87.9%-99.6%) for trisomy 18, and 100% (95%CI 83.2%-100%) for trisomy 13. Specificity was 100% (95% CI 99.87%-100%) for trisomy 21, 99.97% (95%CI 99.81%-99.99%) for trisomy 18, and 99.97% (95%CI 99.81%-99.99%) for trisomy 13. Pooled test failure rate was 1.1%. A direct comparison of microarray- and sequencing-based cfDNA found equivalent test accuracy. Included studies suggest that NIPT using microarray-based cfDNA testing has high sensitivity and specificity for detecting fetal trisomy 21, 18, and 13. However, the evidence base is small and at high risk of bias.

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

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

Application of droplet digital PCR for prenatal screening of Down syndrome

Background: This study was proposed to explore the feasibility of droplet digital PCR (ddPCR) analysis for non-invasive prenatal diagnosis of Down syndrome. Materials and Methods: The authors studied maternal plasma samples from 465 pregnant women carrying euploid and trisomy 21 (T21) fetuses. A methylation-sensitive restriction endonuclease, BstUI, was used to digest the hypomethylated holocarboxylase synthetase (HLCS) gene. The authors quantified digestion-resistant HLCS gene on chromosome 21 and fetal-specific rs6636 SNP allele on chromosome 14 by droplet digital PCR analysis. Maternal plasma DNA analysis was performed by comparing the ratio of hypermethylated HLCS to fetal-specific rs6636 SNP allele between two groups. Results: Using a rs6636 SNP allele on chromosome 14 as the reference marker, the authors analyzed 78 euploid and 28 T21 plasma samples. The ratios of the numbers of positive hypermethylated HLCS and the fetal-specific C allele in the euploid and T21 samples were significantly different ( p p Conclusions: The study demonstrated that ddPCR approach can be applied for prenatal screening of trisomy 21.

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

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

Noninvasive detection of F8 int22h-related inversions and sequence variants in maternal plasma of hemophilia carriers

AbstractDirect detection of F8 and F9 sequence variants in maternal plasma of hemophilia carriers has been demonstrated by microfluidics digital PCR. Noninvasive prenatal assessment of the most clinically relevant group of sequence variants among patients with hemophilia, namely, those involving int22h-related inversions disrupting the F8 gene, poses additional challenges because of its molecular complexity. We investigated the use of droplet digital PCR (ddPCR) and targeted massively parallel sequencing (MPS) for maternal plasma DNA analysis to noninvasively determine fetal mutational status in pregnancies at risk for hemophilia. We designed family-specific ddPCR assays to detect causative sequence variants scattered across the F8 and F9 genes. A haplotype-based approach coupled with targeted MPS was applied to deduce fetal genotype by capturing a 7.6-Mb region spanning the F8 gene in carriers with int22h-related inversions. The ddPCR analysis correctly determined fetal hemophilia status in 15 at-risk pregnancies in samples obtained from 8 to 42 weeks of gestation. There were 3 unclassified samples, but no misclassification. Detailed fetal haplotype maps of the F8 gene region involving int22h-related inversions obtained through targeted MPS enabled correct diagnoses of fetal mutational status in 3 hemophilia families. Our data suggest it is feasible to apply targeted MPS to interrogate maternally inherited F8 int22h-related inversions, whereas ddPCR represents an affordable approach for the identification of F8 and F9 sequence variants in maternal plasma. These advancements may bring benefits for the pregnancy management for carriers of hemophilia sequence variants; in particular, the common F8 int22h-related inversions, associated with the most severe clinical phenotype.

Combined Count- and Size-Based Analysis of Maternal Plasma DNA for Noninvasive Prenatal Detection of Fetal Subchromosomal Aberrations Facilitates Elucidation of the Fetal and/or Maternal Origin of the Aberrations

Noninvasive prenatal detection of fetal subchromosomal copy number aberrations (CNAs) can be achieved through massively parallel sequencing of maternal plasma DNA. However, when a mother herself is a carrier of a CNA, one cannot discern if her fetus has inherited the CNA. In addition, false-positive results would become more prevalent when more subchromosomal regions are analyzed. We used a strategy that combined count- and size-based analyses of maternal plasma DNA for the detection of fetal subchromosomal CNAs in 7 target regions for 10 test cases. For the 5 cases in which CNAs were present only in the fetus, the size-based approach confirmed the aberrations detected by the count-based approach. For the 5 cases in which the mother herself carried an aberration, we successfully deduced that 3 of the fetuses had inherited the aberrations and that the other 2 fetuses had not inherited the aberrations. No false positives were observed in this cohort. Combined count- and size-based analysis of maternal plasma DNA permits the noninvasive elucidation of whether a fetus has inherited a CNA from its mother who herself is a carrier of the CNA. This strategy has the potential to improve the diagnostic specificity of noninvasive prenatal testing.

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.

High Levels of Sample-to-Sample Variation Confound Data Analysis for Non-Invasive Prenatal Screening of Fetal Microdeletions.

Our goal was to test the hypothesis that inter-individual genomic copy number variation in control samples is a confounding factor in the non-invasive prenatal detection of fetal microdeletions via the sequence-based analysis of maternal plasma DNA. The database of genomic variants (DGV) was used to determine the “Genomic Variants Frequency” (GVF) for each 50kb region in the human genome. Whole genome sequencing of fifteen karyotypically normal maternal plasma and six CVS DNA controls samples was performed. The coefficient of variation of relative read counts (cv.RTC) for these samples was determined for each 50kb region. Maternal plasma from two pregnancies affected with a chromosome 5p microdeletion was also sequenced, and analyzed using the GCREM algorithm. We found strong correlation between high variance in read counts and GVF amongst controls. Consequently we were unable to confirm the presence of the microdeletion via sequencing of maternal plasma samples obtained from two sequential affected pregnancies. Caution should be exercised when performing NIPT for microdeletions. It is vital to develop our understanding of the factors that impact the sensitivity and specificity of these approaches. In particular, benign copy number variation amongst controls is a major confounder, and their effects should be corrected bioinformatically.

Clinical performance of non-invasive prenatal testing (NIPT) using targeted cell-free DNA analysis in maternal plasma with microarrays or next generation sequencing (NGS) is consistent across multiple controlled clinical studies.

ObjectiveTo evaluate the clinical performance of non‐invasive prenatal testing for trisomy 21, 18, and 13 using targeted cell‐free DNA (cfDNA) analysis.MethodsTargeted cfDNA analysis using DANSR™ and FORTE™ with microarray quantitation was used to evaluate the risk of trisomy 21, 18, and 13 in blinded samples from 799 singleton, twin, natural, and IVF pregnancies. Subjects either had fetal chromosome evaluation by karyotype, FISH, QF‐PCR, or karyotype for newborns with suspected aneuploidy at birth. The results of targeted cfDNA analysis were compared to clinical genetic testing outcomes to assess clinical performance.ResultsTargeted cfDNA analysis with microarray quantification identified 107/108 trisomy 21 cases (99.1%), 29/30 trisomy 18 cases (96.7%), and 12/12 trisomy 13 cases (100%). The specificity was 100% for all three trisomies. Combining this data with all published clinical performance studies using DANSR/FORTE methodology for greater than 23 000 pregnancies, the sensitivity of targeted cfDNA analysis was calculated to be greater than 99% for trisomy 21, 97% for trisomy 18, and 94% for trisomy 13. Specificity for each trisomy was greater than 99.9%.ConclusionTargeted cfDNA analysis demonstrates consistently high sensitivity and extremely low false positive rates for common autosomal trisomies in pregnancy across quantitation platforms. © 2015 Ariosa Diagnostics Inc. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Performance of antenatal reflex DNA screening for Down's syndrome.

Maternal plasma DNA analysis has a high but imperfect antenatal Down's syndrome screening performance. We aimed to determine the effect of combining DNA testing with current tests. In our modelled screening protocol, women provide two samples, one serum sample for a Combined test, and a plasma sample for a possible DNA test. Women with a Combined test risk above a specified level have a DNA test using the plasma sample without the need to recall them for another sample and counselling (ie. in a reflex manner). Women with a failed DNA test after a second attempt using a fresh plasma sample have an Integrated test. Screening performance was estimated according to the proportion of women reflexed to DNA testing and compared with universal DNA testing. Reflexing 10% of women to a DNA test yields a 91% detection rate (DR) for a 0.025% false-positive rate (FPR) and no failed tests, compared with a 98% DR, 0.2% FPR and a 2.5% test failure rate with universal DNA testing (94% for 0.046% if 20% reflexed). DNA test failure rate has little influence on screening performance Reflex DNA testing substantially reduces the FPR with a relatively small loss in detection compared with universal DNA testing, and reduces patient anxiety by avoiding the recall of women for DNA testing.

Noninvasive prenatal testing by maternal plasma DNA analysis: Current practice and future applications

Prenatal screening of fetal chromosomal aneuploidies and some common genetic diseases is an integral part of antenatal care. Definitive prenatal diagnosis is conventionally achieved by the sampling of fetal genetic material by amniocentesis or chorionic villus sampling. Due to the invasiveness of those procedures, they are associated with a 1 in 200 chance of fetal miscarriage. Hence, researchers have been exploring noninvasive ways to sample fetal genetic material. The presence of cell-free DNA released by the fetus into the circulation of its mother was demonstrated in 1997. Circulating fetal DNA is therefore obtainable through the collection of a blood sample from the pregnant woman without posing any physical harm to the fetus. By analyzing this source of fetal genetic material, researchers have succeeded in developing DNA-based noninvasive tests for the assessment of Down syndrome and single gene diseases. Since the end of 2011, tests for the noninvasive assessment of chromosomal aneuploidies have become commercially available in parts of the world. Recommendations from professional groups have since been made regarding how these tests could be incorporated into the framework of existing prenatal screening programs. More recently, cell-free circulating fetal DNA analysis have been shown to be applicable to the deciphering of the fetal molecular karyotype, genome and methylome. It is envisioned that an increasing number of the noninvasive prenatal tests will become clinically available. The ethical, social and legal implications of the introduction of some of these tests would need to be discussed in the context of different cultures, societal values and the legal framework.

Determination of fetal rhesus d status by maternal plasma DNA analysis.

In this study, we assessed the feasibility of fetal RhD genotyping by analysis of cell-free fetal DNA(cffDNA) extracted from plasma samples of Rhesus (Rh) D-negative pregnant women by using real-time polymerase chain reaction (PCR). Fetal genotyping was performed on 30 RhD-negative women between 9 and 39 weeks of gestation who were referred to us for invasive testing [amniocentesis/chorionic villi sampling (CVS)]. The fetal RHD genotype was determined based on real-time PCR method. Exons 7 and 10 of the RHD and SRY genes were targeted.Among the pregnant women, 12 were carrying male and 17 were carrying female fetuses. Out of 29 pregnant women, 21 had RhD-positive and nine had RhD-negative fetuses. One sample (case 12, whose blood group was found to be AB Rh [+]) was excluded due to controversial results from repeated serological analyses. All prenatal results were in concordance with postnatal RhD status and fetal sex without false- positive or -negative results. Performing real-time PCR on cffDNA showed accurate, efficient and reliable results, allowing rapid and high throughput non invasive determination of fetal sex and RhD status in clinical samples.

Noninvasive Prenatal Determination of Twin Zygosity by Maternal Plasma DNA Analysis

The current methods for distinguishing the zygosities of twins include ultrasound scanning, which is nondefinitive, and amniocentesis, which is invasive. We explored the use of massively parallel sequencing of maternal plasma DNA for the noninvasive prenatal assessment of the zygosities of twin pregnancies. Plasma DNA was extracted from blood collected from 8 women pregnant with twins. Target enrichment and massively parallel sequencing were performed for each plasma DNA library. Apparent fractional fetal DNA concentrations were calculated for multiple genomic regions by determining the ratio of minor to major alleles among single-nucleotide polymorphism sites. Variations in the apparent fractional fetal DNA concentrations between genomic regions were used to infer whether individual fetuses in a twin pair were genotypically different and hence dizygotic. The extent of the variation in the apparent fractional fetal DNA concentration across chromosomes was 0.82-1.35 SDs for monozygotic twin pregnancies and 2.42-4.80 SDs for dizygotic twin pregnancies. The proportions of apparent fractional fetal DNA concentration values that deviated beyond the range expected for stochastic variation were 0.00%-1.93% for monozygotic twin pregnancies and 36.2%-78.1% for dizygotic twin pregnancies. After identifying a pair of twins as likely dizygotic, the method also allowed determination of the fractional fetal DNA concentrations contributed by the individual fetuses of a dizygotic twin pair. Noninvasive prenatal determination of twin zygosity by maternal plasma DNA sequencing is feasible. It is also possible to determine the relative fractional fetal DNA concentrations for each fetus for dizygotic twin pregnancies.

Recent advances in the analysis of fetal nucleic acids in maternal plasma

Noninvasive prenatal diagnosis can be achieved by analyzing cell-free fetal DNA in maternal plasma. The fact that circulating fetal DNA represents only a minor fraction of the DNA that is present in maternal plasma has presented analytical challenges for a number of applications. In this review, we discuss such challenges and how they have been resolved by recent developments in the field. Digital molecular counting methods, such as digital PCR and massively parallel sequencing, have enabled high quantitative precision for maternal plasma DNA analysis. Noninvasive prenatal analysis of monogenic disease mutations has been achieved by identifying small quantitative differences between the mutant and wild-type alleles in maternal plasma. By measuring the small increment in the fractional concentrations of DNA derived from potentially aneuploid chromosomes in maternal plasma, fetal chromosomal aneuploidies have been detected with high diagnostic accuracies. Recently, advances in molecular technologies have enhanced the diagnostic applications of maternal plasma DNA analysis for noninvasive prenatal diagnosis. We foresee that this technology could play an increasingly important role in prenatal investigations.

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.

Prenatal assessment of fetal chromosomal and genetic disorders through maternal plasma DNA analysis

The existence of cell free DNA derived from the fetus in the plasma of pregnant women was first demonstrated in 1997. This discovery offered the possibility of non-invasive sampling of fetal genetic material simply through the collection of a maternal blood sample. Such cell free fetal DNA molecules in the maternal circulation have subsequently been shown to originate from the placenta and could be detected from about 7 weeks of gestation. It has been shown that cell free fetal DNA analysis could offer highly accurate assessment of fetal genotype and chromosomal makeup for some applications. Thus, cell free fetal DNA analysis has been incorporated as a part of prenatal screening programs for the prenatal management of sex-linked and sex-associated diseases, rhesus D incompatibility as well as the prenatal detection of Down's syndrome.Cell free fetal DNA analysis may lead to a change in the way prenatal assessments are made.

Non-invasive prenatal diagnosis of down syndrome: a dream come true

Our group discovered the presence of cell-free fetal DNA in plasma of pregnant women. This finding offered opportunities for the development of non-invasive approaches for prenatal diagnosis. However, fetal DNA co-exists with a large background of maternal DNA and amounts to just some 10% of the total DNA in maternal plasma. Despite the technical challenges, we developed an approach based on massively parallel sequencing for the non-invasive detection of fetal Down syndrome by detecting the small increments in chromosome 21 DNA sequences in maternal plasma. We showed in a study of 750 pregnancies that Down syndrome can be accurately identified by direct analysis of maternal plasma DNA. The method could be further adapted for the prenatal detection of trisomy 18 and trisomy 13. A number of groups have since confirmed our findings. Non-invasive prenatal detection of fetal Down syndrome has since been implemented as a routine service at a number of centres around the world. <h3>Acknowledgement</h3> Supported by the University Grants Committee of the Government of the Hong Kong Special Administrative Region, China, under the Areas of Excellence Scheme (AoE/M-04/ 06), the General Research Fund Scheme of the Hong Kong Research Grants Council (CUHK463109), and an Innovation and Technology Fund (ITS/054/09).

Non-invasive prenatal diagnosis of down syndrome: a dream come true

Our group discovered the presence of cell-free fetal DNA in plasma of pregnant women. This finding offered opportunities for the development of non-invasive approaches for prenatal diagnosis. However, fetal DNA co-exists with a large background of maternal DNA and amounts to just some 10% of the total DNA in maternal plasma. Despite the technical challenges, we developed an approach based on massively parallel sequencing for the non-invasive detection of fetal Down syndrome by detecting the small increments in chromosome 21 DNA sequences in maternal plasma. We showed in a study of 750 pregnancies that Down syndrome can be accurately identified by direct analysis of maternal plasma DNA. The method could be further adapted for the prenatal detection of trisomy 18 and trisomy 13. A number of groups have since confirmed our findings. Non-invasive prenatal detection of fetal Down syndrome has since been implemented as a routine service at a number of centres around the world. <h3>Acknowledgement</h3> Supported by the University Grants Committee of the Government of the Hong Kong Special Administrative Region, China, under the Areas of Excellence Scheme (AoE/M-04/ 06), the General Research Fund Scheme of the Hong Kong Research Grants Council (CUHK463109), and an Innovation and Technology Fund (ITS/054/09).

Noninvasive prenatal diagnosis of hemophilia by microfluidics digital PCR analysis of maternal plasma DNA

AbstractHemophilia is a bleeding disorder with X-linked inheritance. Current prenatal diagnostic methods for hemophilia are invasive and pose a risk to the fetus. Cell-free fetal DNA analysis in maternal plasma provides a noninvasive mean of assessing fetal sex in such pregnancies. However, the disease status of male fetuses remains unknown if mutation-specific confirmatory analysis is not performed. Here we have developed a noninvasive test to diagnose whether the fetus has inherited a causative mutation for hemophilia from its mother. The strategy is based on a relative mutation dosage approach, which we have previously established for determining the mutational status of fetuses for autosomal disease mutations. In this study, the relative mutation dosage method is used to deduce whether a fetus has inherited a hemophilia mutation on chromosome X by detecting whether the concentration of the mutant or wild-type allele is overrepresented in the plasma of heterozygous women carrying male fetuses. We correctly detected fetal genotypes for hemophilia mutations in all of the 12 studied maternal plasma samples obtained from at-risk pregnancies from as early as the 11th week of gestation. This development would make the decision to undertake prenatal testing less traumatic and safer for at-risk families.