Fetal Nucleic Acids in Maternal Plasma Toward the Development of Noninvasive Prenatal Diagnosis of Fetal Chromosomal Aneuploidies
ABSTRACT The discovery of cell-free fetal nucleic acids in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. Over the last few years, a number of approaches have been demonstrated to allow such circulating fetal nucleic acids to be used for the prenatal detection of chromosomal aneuploidies. One such approach involves the enrichment of fetal DNA, such as by size fractionation or by the contro- versial formaldehyde treatment technique. A second approach involves the targeting of fetal-specific nucleic acid molecules, including fetal-specific epigenetic markers and placenta-specific mRNA markers. A third approach involves the development of highly discriminatory quantitative methods for chromosome dosage analysis using digital polymerase chain reaction technology. It is likely that these and other methods yet to be developed would allow noninvasive prenatal diagnosis of chromosomal aneuploidies by maternal plasma nucleic acids to be realized in the near future.
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ABSTRACT: OBJECTIVES: Elevated albumin (ALB) mRNA concentration has been reported in the plasma of patients with liver diseases. The plasma ALB mRNA measurement was shown to be an effective indicator of liver pathologies with superior diagnostic sensitivity and specificity when compared with alanine transaminase (ALT). We hypothesized that serial plasma ALB mRNA analysis would be helpful in the early detection and monitoring of post-liver transplantation complications. DESIGN AND METHODS: One hundred and five blood specimens were collected from 24 post-transplant recipients. Biochemical liver function test profiles and plasma ALB mRNA concentrations were assessed. RESULTS: Over the study period, the health status of 14 recipients (58%) remained stable (Stable group). Their plasma ALB mRNA concentrations remained within a low-concentration range. In contrast, 10 recipients (42%) developed 14 episodes of hepatic complications (Unstable group). The median plasma ALB mRNA concentration of the Unstable group was 6.5-times higher than that of the Stable group. Plasma ALB mRNA concentration was elevated on 13/14 (93%) episodes of the hepatic complications while ALT was elevated only on 8/14 (57%) episodes. CONCLUSIONS: The elevation of plasma ALB mRNA may allow sensitive detection of hepatic complications and monitoring of the clinical course in a dynamic fashion. Serial plasma ALB mRNA measurement is potentially useful for post-liver transplantation management.Clinical biochemistry 04/2013; · 2.02 Impact Factor
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ABSTRACT: The analysis of fetal nucleic acids in maternal blood 13 years ago has led to the initiation of noninvasive methods for the early determination of fetal gender, rhesus D status, and a number of aneuploid disorders and hemoglobinopathies. Subsequently, a comparatively large quantity of fetal DNA and RNA has been demonstrated in amniotic fluid as well as small amounts in premature infant saliva. The DNA and RNA in amniotic fluid has permitted an analysis of core transcriptomes, whilst the DNA and RNA in saliva allows the early detection and treatment monitoring of fetal developmental problems. These aspects are discussed together with the methodology and limits of analysis for noninvasive prenatal diagnosis in predictive, preventive, and personalized medicine.International Journal of Women's Health 01/2013; 5:177-86.
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ABSTRACT: Aim: To assess the accuracy of RHD and RHCE genotyping by real-time PCR, through the analysis of fetal DNA in plasma samples of Rh negative pregnant women. Methods: The study consisted in the collection of two tubes of blood (each with 3 ml) from 19 RhD negative pregnant women that attended the obstetric consultation at the Hospital Distrital of Figueira da Foz, E.P.E. One of the samples was used to perform blood typing and the other was used for fetal DNA extraction. The Rh genotype was determined by real-time PCR with specific primers and Taqman® probes for RHD, RHC, RHE, SRY and GLO genes. Results: In 88.2% of cases (15/17) the RHD genotype was concordant with the phenotyping data, with 1 false-positive result (5.9%) and a precision of 94% (pvalue=0.001; K=76.7%). For the RHC gene, there was concordance in 60% of cases (6/10), with 4 false-negative results (40%) and a precision of 60% (pvalue=0,175; K=31%). Relatively to the RHE gene, the concordance achieved was 100% (10/10), with a precision of also 100% (pvalue=0.002; K=100%). Conclusion: The present study confirms the precision of fetal RHD and RHE genotyping in maternal plasma. Performing a similar study with a superior number of samples could allow the implementation of this non-invasive prenatal diagnostic test in laboratorial and clinical routine, in order to follow pregnancies at risk for developing Hemolytic Disease of the Fetus and Newborn.International Journal of Biomedical Laboratory Science (Accepted for publication). 01/2012; 1.1(2):50-58.
Fetal Nucleic Acids in Maternal Plasma
Toward the Development of Noninvasive Prenatal
Diagnosis of Fetal Chromosomal Aneuploidies
Y.M. Dennis Lo
Centre for Research into Circulating Fetal Nucleic Acids, Li Ka Shing Institute of Health
Sciences, and Department of Chemical Pathology,
The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin,
New Territories, Hong Kong SAR, China
The discovery of cell-free fetal nucleic acids in maternal plasma has opened up new
possibilities for noninvasive prenatal diagnosis. Over the last few years, a number of
approaches have been demonstrated to allow such circulating fetal nucleic acids to
be used for the prenatal detection of chromosomal aneuploidies. One such approach
involves the enrichment of fetal DNA, such as by size fractionation or by the contro-
versial formaldehyde treatment technique. A second approach involves the targeting
of fetal-specific nucleic acid molecules, including fetal-specific epigenetic markers and
placenta-specific mRNA markers. A third approach involves the development of highly
discriminatory quantitative methods for chromosome dosage analysis using digital
polymerase chain reaction technology. It is likely that these and other methods yet to be
developed would allow noninvasive prenatal diagnosis of chromosomal aneuploidies
by maternal plasma nucleic acids to be realized in the near future.
Key words: noninvasive prenatal diagnosis; plasma DNA; plasma RNA; epigenetics;
Prenatal diagnosis is an established part of
modern obstetrics service. However, invasive
techniques, such as amniocentesis, are com-
monly used for the definitive diagnosis of fetal
chromosomal aneuploidies, including trisomy
21. Because of the potential risks associated
with such invasive methods, a long-sought goal
invasive methods that do not carry such a risk.
possibilities for noninvasive prenatal diagno-
sis.1,2As circulating fetal DNA only represents
Address for correspondence: Y.M. Dennis Lo, Department of Chem-
ical Pathology, Room 38061, 1/F Clinical Sciences Building, Prince
of Wales Hospital, 30-32 Ngan Shing Street, Shatin, New Territories,
HongKong SAR,China. Voice: +852-2632-2963; fax:+852-2636-5090.
a mean of ∼3% of the DNA that is present in
maternal plasma,3the use of maternal plasma
DNA for the noninvasive prenatal diagnosis of
fetal chromosomal aneuploidies has presented
considerable technical challenge. However, re-
markable advances have occurred in this area
recently. This review summarizes some of these
Enrichment of Fractional
Fetal DNA Concentrations
One approach involves the enrichment of
the fractional fetal DNA concentrations from
the above-mentioned mean of ∼3% to a level
that is amenable to commonly available an-
alytical strategies. One variant of this ap-
proach has been proposed by Dhallan et al.,
who attempted to enhance the fractional fetal
Ann. N.Y. Acad. Sci. 1137: 140–143 (2008). C ?2008 New York Academy of Sciences.
Lo: Noninvasive Prenatal Diagnosis of Fetal Aneuploidies
DNA concentration by suppressing the ma-
ternal DNA background in maternal plasma
using formaldehyde.4They then applied this
approach to the prenatal detection of Down
The main problem with the
only be reproduced by some,6but not other
An alternative approach is based on the dis-
maternal DNA in maternal plasma.9Thus,
from maternal plasma, it has been demon-
strated that the fractional concentration of fe-
tal DNA can be enhanced.10The main disad-
vantage of this approach is that currently an
electrophoretic methodis used forthesize frac-
tionation of plasma DNA, a procedure that is
relatively labor-intensive and potentially prone
to contamination. It is also unknown whether
this approach would provide the degree of fe-
tal DNA enrichment necessary for the nonin-
vasive prenatal diagnosis of fetal chromosomal
of Fetal-Specific Nucleic
An alternative approach is the targeting of
specific nucleic acid populations in maternal
plasma which are virtually completely fetal-
specific. One such population is fetal-specific
DNA methylation markers.11–14One of these
markers is the SERPINB5 gene, which is hy-
pomethylated in the placenta and hypermethy-
lated in maternal blood cells.12Hypomethy-
lated SERPINB5 can therefore be regarded
as a fetal-specific marker in maternal plasma,
as most of the background maternal DNA
in maternal plasma is probably derived from
hematopoietic cells.15As this gene is located on
allelic ratio analysis of a single nucleotide poly-
morphism (SNP) in the hypomethylated form
of SERPINB5 can be used as a marker of tri-
somy 18 in maternal plasma.16This approach
is called the epigenetic allelic ratio approach
(EAR).16The main disadvantage of this ap-
proach is that it is dependent on bisulfite con-
version, a method that may destroy a large
proportion of the input DNA.17In addition,
the allelic ratio approach can only be used
if the fetus is heterozygous for the SNP con-
cerned. Furthermore, it would be necessary to
search for similar epigenetic changes on the
other chromosomes involved in common ane-
uploidies, such as chromosome 21.18In addi-
tion, because of the limitation in the number of
circulating fetal DNA molecules, if the volume
of plasma used is insufficient, the accuracy of
the method would diminish on account of sta-
tistical sampling errors.16As placenta-specific
epigenetic markers have also been found on
chromosomes not normally involved in fetal
chromosomal aneuploidies (e.g., the RASSF1A
gene on chromosome 3),13it is also potentially
possible to determine the fetal chromosome 21
status by measuring the relative concentrations
of fetal-specific epigenetic markers on chromo-
some 21 to those on one or more reference
An alternative approach to target fetal-
specific nucleic acid species is to use placental-
specific mRNA species.19Through the use of
allelic ratio measurement on placenta-specific
on chromosome 21 for Down syndrome), the
aneuploidy status can be ascertained.20This
approach is called the RNA–SNP allelic ratio
approach, and appears to have a high sensitiv-
ity of 90% and a high specificity of 96.5%.20
Compared with the epigenetic approach, the
advantages of the mRNA approach include
(1) the fact that multiple copies of mRNA are
transcribed from a gene active in the placenta;
and (2) mRNA markers can be readily detected
by reverse transcriptase polymerase chain re-
action (PCR) or other amplification strategies,
without having to involve a more complicated
procedure like bisulfite conversion. Similar to
Annals of the New York Academy of Sciences
the EAR approach, the main limitation of the
RNA–SNP approach is that it would only work
To cover a large proportion, multiple placental
mRNA markers and multiple SNPs would be
High-Precision Analytical Approach
Another approach towards the detection of
fetal chromosomal aneuploidies is the devel-
opment of highly discriminatory quantitative
methods which would allow the detection of a
small excess of nucleic acid sequences from a
goal to be achieved.21For example, it has been
shown that for a DNA sample in which 25% is
derived from the placenta and 75% is derived
from maternal blood cells, digital PCR would
allow the correct classification of the chromo-
some 21 aneuploidy status 97% of the time, as
long as at least 7680 DNA molecules are avail-
able for analysis.21Thus, the work by Lo et al.
provides a set of benchmark parameters docu-
menting the statistical relationship between the
target, the number of nucleic acid molecules,
and the frequencies of correct classification of
the aneuploidy status.21For investigators work-
ing on fetal DNA enrichment techniques,4,10
these parameters would be useful to guide the
degree of fetal DNA enrichment needed. For
workers studying the use of epigenetic16,18or
indicate the number of fetal-specific epigenetic
or mRNA marker molecules needed for robust
ods for conducting digital PCR analysis, such
as microfluidics PCR22and emulsion PCR,23
it is expected that this powerful technology
will have an impact on the clinical application
of noninvasive prenatal diagnosis in the near
After a decade of development, the use of
cell-free fetal nucleic acids in maternal plasma
for the noninvasive prenatal detection of chro-
mosomal aneuploidies has been realized in
principle. A number of alternative approaches
have been shown to be of promise. It is hoped
that these and further approaches yet to be de-
vised will deliver a new generation of tests for
noninvasive prenatal diagnosis over the next
few years, making prenatal investigations safer
for pregnant women and their fetuses.
lence grant from the University Grants Com-
mittee of Hong Kong, the Hong Kong Re-
search Grants Council, the Innovation and
Technology Fund, and the Li Ka Shing
Conflicts of Interest
applications on aspects of the use of fetal nu-
cleic acids in maternal plasma for noninvasive
prenatal diagnosis. Some of these technologies
have been licensed to Sequenom, to which the
author is a consultant.
1. Lo, Y.M.D. et al. 1997. Presence of fetal DNA in
maternal plasma and serum. Lancet 350: 485–487.
3. Lo, Y.M.D. et al. 1998. Quantitative analysis of fetal
DNA in maternal plasma and serum: implications
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4. Dhallan, R. et al. 2004. Methods to increase the per-
centage of free fetal DNA recovered from the mater-
nal circulation. JAMA 291: 1114–1119.
5. Dhallan, R. et al. 2007. A non-invasive test for prena-
tal diagnosis based on fetal DNA present in maternal
blood: a preliminary study. Lancet 369: 474–481.
Lo: Noninvasive Prenatal Diagnosis of Fetal Aneuploidies
6. Benachi, A. et al. 2005. Impact of formaldehyde on
the in vitro proportion of fetal DNA in maternal
plasma and serum. Clin. Chem. 51: 242–244.
of fetal DNA in maternal plasma by formaldehyde
treatment. Clin. Chem. 51: 655–658.
8. Chinnapapagari, S.K. et al. 2005. Treatment of ma-
ternal blood samples with formaldehyde does not
alter the proportion of circulatory fetal nucleic acids
(DNA and mRNA) in maternal plasma. Clin. Chem.
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and fetal DNA in maternal plasma. Clin. Chem. 50:
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JAMA 293: 843–849.
11. Poon, L.L. et al. 2002. Differential DNA methylation
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14. Chan, K.C. et al. 2006. Hypermethylated RASSF1A
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sex-mismatched bone marrow transplantation. Clin.
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