Noninvasive prenatal diagnosis using cell-free fetal nucleic acids in maternal plasma

2011 ◽  
pp. 243-253
Author(s):  
Y. M. Dennis Lo
Keyword(s):  
Prenatal Diagnosis ◽  
Nucleic Acids ◽  
Maternal Plasma ◽  
Noninvasive Prenatal Diagnosis

scholarly journals Noninvasive Prenatal Diagnosis of Fetal Chromosomal Aneuploidies by Maternal Plasma Nucleic Acid Analysis

2008 ◽  
Vol 54 (3) ◽  
pp. 461-466 ◽  
Author(s):  
Y M Dennis Lo ◽  
Rossa W K Chiu
Keyword(s):  
Prenatal Diagnosis ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Maternal Plasma ◽  
Small Sample ◽  
Routine Practice ◽  
Allelic Ratio ◽  
Noninvasive Prenatal Diagnosis ◽  
Nucleic Acid Analysis ◽  
Chromosomal Aneuploidies

Abstract Background: The discovery of circulating cell-free fetal nucleic acids in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. The potential application of this technology for the noninvasive prenatal detection of fetal chromosomal aneuploidies is an aspect of this field that is being actively investigated. The main challenge of work in this area is the fact that cell-free fetal nucleic acids represent only a minor fraction of the total nucleic acids in maternal plasma. Methods and Results: We performed a review of the literature, which revealed that investigators have applied methods based on the physical and molecular enrichment of fetal nucleic acid targets from maternal plasma. The former includes the use of size fractionation of plasma DNA and the use of the controversial formaldehyde treatment method. The latter has been achieved through the development of fetal epigenetic and fetal RNA markers. The aneuploidy status of the fetus has been explored through the use of allelic ratio analysis of plasma fetal epigenetic and RNA markers. Digital PCR has been shown to offer high precision for allelic ratio and relative chromosome dosage analyses. Conclusions: After a decade of work, the theoretical and practical feasibility of prenatal fetal chromosomal aneuploidy detection by plasma nucleic acid analysis has been demonstrated in studies using small sample sets. Larger scale independent studies will be needed to validate these initial observations. If these larger scale studies prove successful, it is expected that with further development of new fetal DNA/RNA markers and new analytical methods, molecular noninvasive prenatal diagnosis of the major chromosomal aneuploidies could become a routine practice in the near future.

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 Noninvasive Prenatal Diagnosis of Single-Gene Disorders by Use of Droplet Digital PCR

2018 ◽  
Vol 64 (2) ◽  
pp. 336-345 ◽  
Author(s):  
Joan Camunas-Soler ◽  
Hojae Lee ◽  
Louanne Hudgins ◽  
Susan R Hintz ◽  
Yair J Blumenfeld ◽  
...  
Keyword(s):  
At Risk ◽  
Prenatal Diagnosis ◽  
Metabolic Disorders ◽  
Single Gene ◽  
Digital Pcr ◽  
Maternal Plasma ◽  
Single Nucleotide ◽  
Noninvasive Prenatal Diagnosis ◽  
Single Gene Disorders ◽  
Fetal Fraction

Abstract BACKGROUND Prenatal diagnosis in pregnancies at risk of single-gene disorders is currently performed using invasive methods such as chorionic villus sampling and amniocentesis. This is in contrast with screening for common aneuploidies, for which noninvasive methods with a single maternal blood sample have become standard clinical practice. METHODS We developed a protocol for noninvasive prenatal diagnosis of inherited single-gene disorders using droplet digital PCR from circulating cell-free DNA (cfDNA) in maternal plasma. First, the amount of cfDNA and fetal fraction is determined using a panel of TaqMan assays targeting high-variability single-nucleotide polymorphisms. Second, the ratio of healthy and diseased alleles in maternal plasma is quantified using TaqMan assays targeting the mutations carried by the parents. Two validation approaches of the mutation assay are presented. RESULTS We collected blood samples from 9 pregnancies at risk for different single-gene disorders, including common conditions and rare metabolic disorders. We measured cases at risk of hemophilia, ornithine transcarbamylase deficiency, cystic fibrosis, β-thalassemia, mevalonate kinase deficiency, acetylcholine receptor deficiency, and DFNB1 nonsyndromic hearing loss. We correctly differentiated affected and unaffected pregnancies (2 affected, 7 unaffected), confirmed by neonatal testing. We successfully measured an affected pregnancy as early as week 11 and with a fetal fraction as low as 3.7% (0.3). CONCLUSIONS Our method detects single-nucleotide mutations of autosomal recessive diseases as early as the first trimester of pregnancy. This is of importance for metabolic disorders in which early diagnosis can affect management of the disease and reduce complications and anxiety related to invasive testing.

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.

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