Noninvasive prenatal diagnosis of congenital adrenal hyperplasia in cell-free fetal DNA with COLD-PCR

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.

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

Clinical Chemistry ◽

10.1373/clinchem.2006.076851 ◽

2006 ◽

Vol 52 (12) ◽

pp. 2194-2202 ◽

Cited By ~ 117

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.

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Recent Advances in Fetal Nucleic Acids in Maternal Plasma

Journal of Histochemistry & Cytochemistry ◽

10.1369/jhc.4r6362.2005 ◽

2005 ◽

Vol 53 (3) ◽

pp. 293-296 ◽

Cited By ~ 36

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.

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Applications of Cell-Free Fetal DNA in Maternal Serum

International Journal of Infertility & Fetal Medicine ◽

10.5005/jp-journals-10016-1038 ◽

2012 ◽

Vol 3 (2) ◽

pp. 33-39 ◽

Cited By ~ 1

Author(s):

Saeid Ghorbian

Keyword(s):

Prenatal Diagnosis ◽

Single Gene ◽

Late Pregnancy ◽

Maternal Serum ◽

Maternal Circulation ◽

Fetal Dna ◽

Noninvasive Prenatal Diagnosis ◽

Cell Free Fetal Dna ◽

Paternity Determination ◽

Free Circulating Dna

ABSTRACT Cell-free fetal DNA (cffDNA) is available in the maternal circulation throughout pregnancy and can be used for noninvasive prenatal diagnosis including, determination of fetal sex, identification of specific single gene disorders, typing of fetal blood groups (RhD), paternity determination and potentially routine use for Down's syndrome (DS) testing of all pregnancies. I searched published literature on the PubMed and databases on Scopus interface systematically using keyword's cffDNA, noninvasive diagnosis, fetal DNA in the maternal serum. Reference lists from the papers were also searched. cffDNA representing only 3% of the total cell-free circulating DNA in early and rising to 12% in late pregnancy, clinical investigations has already demonstrated the potential advantage, such as improving safety, earlier diagnosis and comparative ease of testing using cffDNA technology. The discovery of cffDNA circulating in the maternal serum has opened the door to noninvasive prenatal diagnosis testing with novel clinical implications. How to cite this article Ghorbian S. Applications of Cell-Free Fetal DNA in Maternal Serum. Int J Infertility Fetal Med 2012;3(2):33-39.

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