scholarly journals Size Distributions of Maternal and Fetal DNA in Maternal Plasma

2004 ◽  
Vol 50 (1) ◽  
pp. 88-92 ◽  
Author(s):  
K C Allen Chan ◽  
Jun Zhang ◽  
Angela B Y Hui ◽  
Nathalie Wong ◽  
Tze K Lau ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Size Distribution ◽  
Maternal Plasma ◽  
Rapid Expansion ◽  
Molecular Characteristics ◽  
Dna Fragments ◽  
Dna Molecules ◽  
Plasma Dna ◽  
Fetal Dna ◽  
Noninvasive Prenatal Diagnosis

Abstract Background: The discovery of fetal DNA in maternal plasma has opened up an approach for noninvasive prenatal diagnosis. Despite the rapid expansion in clinical applications, the molecular characteristics of plasma DNA in pregnant women remain unclear. Methods: We investigated the size distribution of plasma DNA in 34 nonpregnant women and 31 pregnant women, using a panel of quantitative PCR assays with different amplicon sizes targeting the leptin gene. We also determined the size distribution of fetal DNA in maternal plasma by targeting the SRY gene. Results: The median percentages of plasma DNA with size >201 bp were 57% and 14% for pregnant and nonpregnant women, respectively (P <0.001, Mann–Whitney test). The median percentages of fetal-derived DNA with sizes >193 bp and >313 bp were 20% and 0%, respectively, in maternal plasma. Conclusion: Plasma DNA molecules are mainly short DNA fragments. The DNA fragments in the plasma of pregnant women are significantly longer than those in the plasma of nonpregnant women, and the maternal-derived DNA molecules are longer than the fetal-derived ones.

scholarly journals Noninvasive Prenatal Diagnosis of Monogenic Diseases by Targeted Massively Parallel Sequencing of Maternal Plasma: Application to β-Thalassemia

2012 ◽  
Vol 58 (10) ◽  
pp. 1467-1475 ◽  
Author(s):  
Kwan-Wood G Lam ◽  
Peiyong Jiang ◽  
Gary J W Liao ◽  
K C Allen Chan ◽  
Tak Y Leung ◽  
...  
Keyword(s):  
Prenatal Diagnosis ◽  
Massively Parallel Sequencing ◽  
Globin Gene ◽  
Maternal Plasma ◽  
Massively Parallel ◽  
Sequencing Data ◽  
Plasma Dna ◽  
Parallel Sequencing ◽  
Noninvasive Prenatal Diagnosis ◽  
Monogenic Diseases

Abstract BACKGROUND A genomewide genetic and mutational profile of a fetus was recently determined via deep sequencing of maternal plasma DNA. This technology could have important applications for noninvasive prenatal diagnosis (NIPD) of many monogenic diseases. Relative haplotype dosage (RHDO) analysis, a core step of this procedure, would allow one to elucidate the maternally inherited half of the fetal genome. For clinical applications, the cost and complexity of data analysis might be reduced via targeted application of this approach to selected genomic regions containing disease-causing genes. There is thus a need to explore the feasibility of performing RHDO analysis in a targeted manner. METHODS We performed target enrichment by using solution-phase hybridization followed by massively parallel sequencing of the β-globin gene region in 2 families undergoing prenatal diagnosis for β-thalassemia. We used digital PCR strategies to physically deduce parental haplotypes. Finally, we performed RHDO analysis with target-enriched sequencing data and parental haplotypes to reveal the β-thalassemic status for the fetuses. RESULTS A mean sequencing depth of 206-fold was achieved in the β-globin gene region by targeted sequencing of maternal plasma DNA. RHDO analysis was successful for the sequencing data obtained from the target-enriched samples, including a region in one of the families in which the parents had similar haplotype structures. Data analysis revealed that both fetuses were heterozygous carriers of β-thalassemia. CONCLUSIONS Targeted sequencing of maternal plasma DNA for NIPD of monogenic diseases is feasible.

scholarly journals Microsystem for Isolation of Fetal DNA from Maternal Plasma by Preparative Size Separation

2009 ◽  
Vol 55 (12) ◽  
pp. 2144-2152 ◽  
Author(s):  
Thomas Hahn ◽  
Klaus S Drese ◽  
Ciara K O'Sullivan
Keyword(s):  
Prenatal Diagnosis ◽  
High Volume ◽  
Maternal Plasma ◽  
Chromosomal Anomalies ◽  
Size Separation ◽  
Large Samples ◽  
Fetal Dna ◽  
Noninvasive Prenatal Diagnosis ◽  
Cell Free Fetal Dna ◽  
Invasive Techniques

Abstract Background: Routine prenatal diagnosis of chromosomal anomalies is based on invasive procedures, which carry a risk of approximately 1%–2% for loss of pregnancy. An alternative to these inherently invasive techniques is to isolate fetal DNA circulating in the pregnant mother’s plasma. Free fetal DNA circulates in maternal plasma primarily as fragments of lengths <500 bp, with a majority being <300 bp. Separating these fragments by size facilitates an increase in the ratio of fetal to maternal DNA. Methods: We describe our development of a microsystem for the enrichment and isolation of cell-free fetal DNA from maternal plasma. The first step involves a high-volume extraction from large samples of maternal plasma. The resulting 80-μL eluate is introduced into a polymeric microsystem within which DNA is trapped and preconcentrated. This step is followed by a transient isotachophoresis step in which the sample stacks within a neighboring channel for subsequent size separation and is recovered via an outlet at the end of the channel. Results: Recovered fractions of fetal DNA were concentrated 4–8 times over those in preconcentration samples. With plasma samples from pregnant women, we detected the fetal SRY gene (sex determining region Y) exclusively in the fragment fraction of <500 bp, whereas a LEP gene (leptin) fragment was detected in both the shorter and longer recovery fractions. Conclusions: The microdevice we have described has the potential to open new perspectives in noninvasive prenatal diagnosis by facilitating the isolation of fetal DNA from maternal plasma in an integrated, inexpensive, and easy-to-use microsystem.

scholarly journals Noninvasive Prenatal Detection of Trisomy 21 by an Epigenetic–Genetic Chromosome-Dosage Approach

2010 ◽  
Vol 56 (1) ◽  
pp. 90-98 ◽  
Author(s):  
Yu K Tong ◽  
Shengnan Jin ◽  
Rossa WK Chiu ◽  
Chunming Ding ◽  
KC Allen Chan ◽  
...  
Keyword(s):  
Prenatal Diagnosis ◽  
Genetic Markers ◽  
Blood Cells ◽  
Trisomy 21 ◽  
Maternal Blood ◽  
Maternal Plasma ◽  
Plasma Samples ◽  
Prenatal Detection ◽  
Fetal Dna ◽  
Noninvasive Prenatal Diagnosis

Abstract Background: The use of fetal DNA in maternal plasma for noninvasive prenatal diagnosis of trisomy 21 (T21) is an actively researched area. We propose a novel method of T21 detection that combines fetal-specific epigenetic and genetic markers. Methods: We used combined bisulfite restriction analysis to search for fetal DNA markers on chromosome 21 that were differentially methylated in the placenta and maternal blood cells and confirmed any target locus with bisulfite sequencing. We then used methylation-sensitive restriction endonuclease digestion followed by microfluidics digital PCR analysis to investigate the identified marker. Chromosome-dosage analysis was performed by comparing the dosage of this epigenetic marker with that of the ZFY (zinc finger protein, Y-linked) gene on chromosome Y. Results: The putative promoter of the HLCS (holocarboxylase synthetase) gene was hypermethylated in the placenta and hypomethylated in maternal blood cells. A chromosome-dosage comparison of the hypermethylated HLCS and ZFY loci could distinguish samples of T21 and euploid placental DNA. Twenty-four maternal plasma samples from euploid pregnancies and 5 maternal plasma samples from T21 pregnancies were analyzed. All but 1 of the euploid samples were correctly classified. Conclusions: The epigenetic–genetic chromosome-dosage approach is a new method for noninvasive prenatal detection of T21. The epigenetic part of the analysis can be applied to all pregnancies. Because the genetic part of the analysis uses paternally inherited, fetal-specific genetic markers that are abundant in the genome, broad population coverage should be readily achievable. This approach has the potential to become a generally usable technique for noninvasive prenatal diagnosis.

scholarly journals Haplotype-Based Noninvasive Prenatal Diagnosis for Duchenne Muscular Dystrophy: A pilot study in South China

2019 ◽  
Author(s):  
Min Chen ◽  
Chao Chen ◽  
Yingting Li ◽  
Yuan Yuan ◽  
Zhengfei Lai ◽  
...  
Keyword(s):  
Prenatal Diagnosis ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Maternal Plasma ◽  
Targeted Sequencing ◽  
Plasma Dna ◽  
Noninvasive Prenatal Diagnosis ◽  
Integration Analysis ◽  
Dependent Probe ◽  
Singleton Pregnancies

AbstractObjectiveTo explore the accuracy and feasibility of noninvasive prenatal diagnosis (NIPD) for Duchenne Muscular Dystrophy (DMD) based on the haplotype approach.MethodsWe recruited singleton pregnancies at-risk of DMD at 12-25 weeks of gestation from 17 families who all had a proband children affected by DMD. We have identified the pathogenic mutations in probands and their mothers by multiplex ligation-dependent probe amplification (MLPA). To construct parental haplotypes, we performed captured sequencing on genomic DNA from parents and probands. The integration analysis of parental haplotypes and targeted sequencing results of maternal plasma DNA were used to infer the fetal haplotype and genotypes in DMD gene. Fetal DMD genotypes were further confirmed by invasive prenatal diagnosis.ResultsWe have successfully performed the haplotype-based NIPD in all recruited families. Ten fetuses were identified as normal, including four female and six male fetuses. Four female fetuses were carriers and the other three male fetuses were affected by DMD with exons 49-52 deletion, exons 8-37 deletion and c.628G > T mutation, respectively. The results of NIPD were consistent with those of invasive diagnosis.ConclusionHaplotype-based NIPD for DMD by targeted sequencing is promising and has potential for clinical application.

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

2021 ◽  
Vol 118 (50) ◽  
pp. e2114937118
Author(s):  
Stephanie C. Y. Yu ◽  
Peiyong Jiang ◽  
Wenlei Peng ◽  
Suk Hang Cheng ◽  
Y. T. Tommy Cheung ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Analysis ◽  
Large Population ◽  
Maternal Plasma ◽  
Dna Molecules ◽  
Plasma Dna ◽  
Cell Free Dna ◽  
Dna Molecule ◽  
Free Dna ◽  
Monogenic Diseases

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.

scholarly journals Noninvasive Prenatal Detection of Fetal Trisomy 18 by Epigenetic Allelic Ratio Analysis in Maternal Plasma: Theoretical and Empirical Considerations

2006 ◽  
Vol 52 (12) ◽  
pp. 2194-2202 ◽  
Author(s):  
Yu K Tong ◽  
Chunming Ding ◽  
Rossa WK Chiu ◽  
Ageliki Gerovassili ◽  
Stephen SC Chim ◽  
...  
Keyword(s):  
Prenatal Diagnosis ◽  
Maternal Plasma ◽  
Trisomy 18 ◽  
Ratio Analysis ◽  
Dna Mixtures ◽  
Allelic Ratio ◽  
Fetal Dna ◽  
Noninvasive Prenatal Diagnosis ◽  
Cell Free Fetal Dna ◽  
Chromosomal Aneuploidies

Abstract Background: The discovery of cell-free fetal DNA in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. However, the use of maternal plasma fetal DNA for the direct detection of fetal chromosomal aneuploidies has not been reported. We postulate that the aneuploidy status of a fetus could be revealed by an epigenetic allelic ratio approach, i.e., by analyzing the allelic ratio of a single-base variation present within DNA molecules exhibiting a placental-specific epigenetic signature in maternal plasma. Methods: Placental-derived fetal-specific unmethylated maspin (SERPINB5) promoter sequences on human chromosome 18 were detectable in placental–maternal DNA mixtures and in maternal plasma by bisulfite modification followed by methylation-specific PCR (MSP) and primer extension. The ratios between the extension products of the 2 alleles were calculated for heterozygous placentas, placental–maternal blood cell DNA mixtures, and maternal plasma samples. The allelic ratios were compared between pregnancies carrying trisomy 18 and euploid fetuses. Results: The epigenetic allelic ratios of all tested trisomy 18 samples deviated from the reference range obtained from euploid samples (placental DNA, 1.135 to 2.052; placental–maternal DNA mixtures, 1.170 to 1.985; maternal plasma, 0.330 to 3.044; without skew correction on the raw mass spectrometric data). A theoretical model was established and validated that predicted that a minimum of 200 copies of genomic DNA after bisulfite conversion were required for distinguishing euploid and aneuploid fetuses with confidence. Conclusion: Epigenetic allelic ratio analysis of maternal plasma DNA represents a promising approach for noninvasive prenatal diagnosis of fetal chromosomal aneuploidies.

scholarly journals Recent Advances in Fetal Nucleic Acids in Maternal Plasma

2005 ◽  
Vol 53 (3) ◽  
pp. 293-296 ◽  
Author(s):  
Y.M. Dennis Lo
Keyword(s):  
Prenatal Diagnosis ◽  
First Trimester ◽  
Thalassemia Major ◽  
Maternal Plasma ◽  
Fetal Dna ◽  
Noninvasive Prenatal Diagnosis ◽  
Cell Free Fetal Dna ◽  
Circulating Fetal Dna ◽  
Circulating Rna ◽  
Near Future

The discovery of cell-free fetal DNA in maternal plasma in 1997 has opened up new possibilities for noninvasive prenatal diagnosis. Circulating fetal DNA molecules have been detected in maternal plasma from the first trimester onwards and can be robustly detected using a variety of molecular methods. This approach has been used for the prenatal investigation of sex-linked diseases, fetal RhD status, and prenatal exclusion of β-thalassemia major. Recently, fetal RNA has also been found in maternal plasma. Such fetal RNA has been shown to originate from the placenta and to be remarkably stable. The use of microarray-based approaches has made it feasible to rapidly generate new circulating RNA markers. It is hoped that further developments in this field will make the routine and widespread practice of noninvasive nucleic acid-based prenatal diagnosis for common pregnancy-associated disorders feasible in the near future.

scholarly journals Maternal Plasma DNA Analysis with Massively Parallel Sequencing by Ligation for Noninvasive Prenatal Diagnosis of Trisomy 21

2010 ◽  
Vol 56 (3) ◽  
pp. 459-463 ◽  
Author(s):  
Rossa WK Chiu ◽  
Hao Sun ◽  
Ranjit Akolekar ◽  
Christopher Clouser ◽  
Clarence Lee ◽  
...  
Keyword(s):  
Prenatal Diagnosis ◽  
Trisomy 21 ◽  
Massively Parallel Sequencing ◽  
Maternal Plasma ◽  
Chromosome 21 ◽  
Massively Parallel ◽  
Plasma Dna ◽  
Parallel Sequencing ◽  
Noninvasive Prenatal Diagnosis ◽  
Sequencing By Synthesis

Abstract Background: Noninvasive prenatal diagnosis of trisomy 21 (T21) has recently been shown to be achievable by massively parallel sequencing of maternal plasma on a sequencing-by-synthesis platform. The quantification of several other human chromosomes, including chromosomes 18 and 13, has been shown to be less precise, however, with quantitative biases related to the chromosomal GC content. Methods: Maternal plasma DNA from 10 euploid and 5 T21 pregnancies was sequenced with a sequencing-by-ligation approach. We calculated the genomic representations (GRs) of sequenced reads from each chromosome and their associated measurement CVs and compared the GRs of chromosome 21 (chr21) for the euploid and T21 pregnancies. Results: We obtained a median of 12 × 106 unique reads (21% of the total reads) per sample. The GRs deviated from those expected for some chromosomes but in a manner different from that previously reported for the sequencing-by-synthesis approach. Measurements of the GRs for chromosomes 18 and 13 were less precise than for chr21. z Scores of the GR of chr21 were increased in the T21 pregnancies, compared with the euploid pregnancies. Conclusions: Massively parallel sequencing-by-ligation of maternal plasma DNA was effective in identifying T21 fetuses noninvasively. The quantitative biases observed among the GRs of certain chromosomes were more likely based on analytical factors than biological factors. Further research is needed to enhance the precision for measuring for the representations of chromosomes 18 and 13.

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