Non-invasive epigenetic detection of fetal trisomy 21 in first trimester maternal plasma.

Abstract

Down syndrome (DS) is the most common known aneuploidy, caused by an extra copy of all or part of chromosome 21. Fetal-specific epigenetic markers have been investigated for non-invasive prenatal detection of fetal DS. The phosphodiesterases gene, PDE9A, located on chromosome 21q22.3, is completely methylated in blood (M-PDE9A) and unmethylated in the placenta (U-PDE9A). Therefore, we estimated the accuracy of non-invasive fetal DS detection during the first trimester of pregnancy using this tissue-specific epigenetic characteristic of PDE9A.
A nested, case-control study was conducted using maternal plasma samples collected from 108 pregnant women carrying 18 DS and 90 normal fetuses (each case was matched with 5 controls according to gestational weeks at blood sampling). All pregnancies were singletons at or before 12 weeks of gestation between October 2008 and May 2009. The maternal plasma levels of M-PDE9A and U-PDE9A were measured by quantitative methylation-specific polymerase chain reaction. M-PDE9A and U-PDE9A levels were obtained in all samples and did not differ between male and female fetuses. M-PDE9A levels did not differ between the DS cases and controls (1854.3 vs 2004.5 copies/mL; P = 0.928). U-PDE9A levels were significantly elevated in women with DS fetuses compared with controls (356.8 vs 194.7 copies/mL, P<0.001). The sensitivities of U-PDE9A level and the unmethylation index of PDE9A for non-invasive fetal DS detection were 77.8% and 83.3%, respectively, with a 5% false-positive rate. In the risk assessment for fetal DS, the adjusted odds ratios of U-PDE9A level and UI were 46.2 [95% confidence interval: 7.8-151.6] and 63.7 [95% confidence interval: 23.2-206.7], respectively.
Our findings suggest that U-PDE9A level and the unmethylation index of PDE9A may be useful biomarkers for non-invasive fetal DS detection during the first trimester of pregnancy, regardless of fetal gender.

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Non-Invasive Epigenetic Detection of Fetal Trisomy 21 in
First Trimester Maternal Plasma
Ji Hyae Lim1, Shin Young Kim1, So Yeon Park1, Shin Yeong Lee1, Mi Jin Kim1, You Jung Han2, Si Won
Lee2, Jin Hoon Chung2, Moon Young Kim2, Jae Hyug Yang2, Hyun Mee Ryu1,2*
1Laboratory of Medical Genetics, Medical Research Institute, Cheil General Hospital and Women’s Healthcare Center, Seoul, Korea, 2Department of Obstetrics and
Gynecology, Cheil General Hospital and Women’s Healthcare Center, KwanDong University college of Medicine, Seoul, Korea
Abstract
Background: Down syndrome (DS) is the most common known aneuploidy, caused by an extra copy of all or part of
chromosome 21. Fetal-specific epigenetic markers have been investigated for non-invasive prenatal detection of fetal DS.
The phosphodiesterases gene, PDE9A, located on chromosome 21q22.3, is completely methylated in blood (M-PDE9A) and
unmethylated in the placenta (U-PDE9A). Therefore, we estimated the accuracy of non-invasive fetal DS detection during the
first trimester of pregnancy using this tissue-specific epigenetic characteristic of PDE9A.
Methodology/Principal Findings: A nested, case-control study was conducted using maternal plasma samples collected
from 108 pregnant women carrying 18 DS and 90 normal fetuses (each case was matched with 5 controls according to
gestational weeks at blood sampling). All pregnancies were singletons at or before 12 weeks of gestation between October
2008 and May 2009. The maternal plasma levels of M-PDE9A and U-PDE9A were measured by quantitative methylation-
specific polymerase chain reaction. M-PDE9A and U-PDE9A levels were obtained in all samples and did not differ between
male and female fetuses. M-PDE9A levels did not differ between the DS cases and controls (1854.3 vs 2004.5 copies/mL;
P=0.928). U-PDE9A levels were significantly elevated in women with DS fetuses compared with controls (356.8 vs
194.7 copies/mL, P,0.001). The sensitivities of U-PDE9A level and the unmethylation index of PDE9A for non-invasive fetal
DS detection were 77.8% and 83.3%, respectively, with a 5% false-positive rate. In the risk assessment for fetal DS, the
adjusted odds ratios of U-PDE9A level and UI were 46.2 [95% confidence interval: 7.8–151.6] and 63.7 [95% confidence
interval: 23.2–206.7], respectively.
Conclusions: Our findings suggest that U-PDE9A level and the unmethylation index of PDE9A may be useful biomarkers for
non-invasive fetal DS detection during the first trimester of pregnancy, regardless of fetal gender.
Citation: Lim JH, Kim SY, Park SY, Lee SY, Kim MJ, et al. (2011) Non-Invasive Epigenetic Detection of Fetal Trisomy 21 in First Trimester Maternal Plasma. PLoS
ONE 6(11): e27709. doi:10.1371/journal.pone.0027709
Editor: Qamaruddin Nizami, Aga Khan University, Pakistan
Received July 24, 2011; Accepted October 23, 2011; Published November 23, 2011
Copyright: ? 2011 Lim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by a research grant from the Life Insurance Philanthropy Foundation. The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: hmryu@yahoo.com
Introduction
Prenatal testing is an integral component of obstetric practice.
Detection of chromosomal aneuploidy has been considered the
most common and important aspect of prenatal testing. Aneu-
ploidy refers to changes in the numbers of chromosomes present
inside a cell, and phenotypic effects are caused by an increased or
decreased gene dosage as a result of gaining or losing a particular
chromosome. The most common aneuploidy is trisomy 21, refered
to as Down syndrome (DS), which occurs in 1 in 800 live births
worldwide and is caused by an extra copy of all or part of
chromosome 21 [1]. DS is associated with intellectual impairment,
severe learning difficulties, and mortality caused by long-term
health problems such as heart disease [1]. The incidence of fetal
DS increases in a maternal age-dependent manner [1,2].
Worldwide, millions of pregnant women undergo prenatal
testing including screening tests and invasive diagnostic procedures
for fetal DS detection every year. The current screening protocol
for fetal DS involves a blood test for maternal protein biomarkers
associated with DS (maternal serum alpha fetoprotein, unconju-
gated estriol, human chorionic gonadotrophin, and inhibin-A) as
well as fetal ultrasound evaluations of nuchal translucency.
However, the detection rate for each factor ranges from 25% to
68% with false-positive rate of 5% [3,4]. Therefore, multiple
screening tests are usually performed to increase the detection rate
for fetal DS at 11–12 weeks and 16–18 weeks of gestation.
However, such integrated tests have disadvantages such as
increased cost due to the use of multiple biomarkers and
psychological burden on the patient as they wait to obtain results
in the second trimester (16–18 weeks of gestation). Moreover, 5%
of normal pregnant women with false-positive results undergo
unnecessary invasive diagnostic processes such as amniocentesis or
chorionic villus sampling (CVS) for fetal DS detection. Such
invasive diagnostic tests are expensive, require expert technicians,
and carry a risk of miscarriage of approximately 1% [5,6].
Therefore, non-invasive prenatal test that allow for the direct
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analysis of fetal genetic material in maternal blood have been
investigated as alternative methods to reduce the need for invasive
procedures and to improve the efficacy of current screening tests
for fetal DS.
Cyclic nucleotide phosphodiesterases (PDEs) catalyze the
hydrolysis of cyclic adenosine 30,50-monophosphate (cAMP)
and/or cyclic guanosine 30,50-monophosphate (cGMP) [7]. The
human PDE9A gene, located on chromosome 21q22.3, encodes a
cGMP-specific PDE that is expressed in most tissues, but not in
blood [8,9]. Due to its location and contribution to the regulation
of the steady-state cellular concentrations of cyclic nucleotides,
PDE9A is a possible candidate gene for diseases such as bipolar
affective disorder [9], and Guipponi et al. suggested that its
overexpression might be involved in DS [9]. Recently, Chim et al.
performed a systematic search for fetal-specific epigenetic markers
on chromosome 21 [10], during which they found that the PED9A
region is completely methylated in the maternal blood (M-PED9A)
and unmethylated in fetal (placental) tissues (U-PED9A). There-
fore, they reported that this marker was independent of fetal
gender and genotype, and hence could be applied to all fetal-
maternal pairs for non-invasive detection of fetal sequences on
chromosome 21. However, the usefulness of PED9A in the non-
invasive prenatal detection of fetal DS has not yet been clinically
validated.
The aim of this study was to estimate the accuracy of non-
invasive fetal DS detection using tissue specific-methylation in
PDE9A and to evaluate the potential of PDE9A as a new biomarker
for the first trimester detection of fetal DS.
Materials and Methods
Ethics statement
This study was conducted according to the principles expressed
in the Declaration of Helsinki. Appropriate institutional review
board approval was obtained from the Ethics Committee at Cheil
General Hospital for this study (#CGH-IRB-2008-43). Written
informed consent was obtained from each participant before blood
draws for the collection of samples and subsequent analysis.
Study participants and samples
We performed a nested case-control study of women who
enrolled in the Cheil General Hospital Non-invasive Prenatal
Diagnosis Study (CNPD). Participants in the CNPD were
recruited from 2008 for non-invasive prenatal diagnosis of rare
and incurable fetal diseases. Participants were women who
received prenatal care at Cheil General Hospital. For the current
study, 108 women were selected from a larger sample of 793
women enrolled in the CNPD according to criteria described
below. All pregnancies were singletons at or before 12 weeks of
gestation between October 2008 and May 2009.
The case group consisted of 18 women who were subsequently
detected to be carrying a DS fetus by amniocentesis or CVS for
fetal karyotyping. Each case was paired with five controls that
were matched according to gestational week at blood sampling
(Table S1). The control group (n=90) included women who
delivered healthy normal neonates at term (37 weeks of gestation
or more) without medical or obstetric complications, such as
hypertension, diabetes, renal insufficiency, congenital anomalies,
or fetal demise. Before maternal blood sampling, ultrasonogra-
phy was recommended to establish the viability of each singleton
pregnancy and to confirm gestational age calculated from the
time of last menstruation. Maternal, fetal, and infant records
were collected prospectively and maintained in an electronic
database. None of the participants had histories of preexisting
hypertension, diabetes mellitus, liver disease, or chronic kidney
disease.
Isolation of Plasma and DNA
Ten milliliters of peripheral blood were collected from each
participant into ethylenediaminetetraacetic acid (EDTA) tubes.
Immediately after sampling, maternal peripheral blood samples
were centrifuged at 1,600 g for 10 minutes at 4uC and the plasma
portion was recentrifuged at 16,000 g for 10 minutes to minimize
any additional release of maternal DNA. Circulating fetal DNA
was extracted from 1.5 mL of maternal plasma using the QIAamp
DSP Virus Kit (Qiagen, Hilden, Germany), according to the
manufacturer’s recommendations. Each DNA sample was eluted
into 50 mL of sterile, DNase-free water. The samples were coded
for subsequent blinded analysis.
Quantitative methylation-specific polymerase chain
reaction (qMSP)
Differences in methylation of PDE9A in maternal blood cells
and placenta were confirmed by bisulphite genomic sequencing
(Fig. 1, Methods S1). Levels of M-PDE9A and U-PDE9A in
maternal plasma were measured by qMSP assays as previously
described [10,11].
DNA extracted from plasma was bisulphite-converted using
the EZ DNA Methylation Kit (Zymo Research, Orange, CA,
USA) and then eluted into 30 mL. For the validation of bisulphite
conversion, a synthetic DNA oligonucleotide for the U-PDE9A
region and genomic DNA extracted from peripheral blood cells
were used as positive and negative controls, respectively. The
PCR reaction solution contained 12.5 mL iQ Supermix (Bio-Rad,
Hercules, CA, USA), 200 nM hydrolysis probe (Applied Biosys-
tems, Foster City, CA, USA), 400 nM primers (Applied
Biosystems, Foster City, CA, USA), and 5 mL converted DNA
per 25 mL total reaction volume. The primers and hydrolysis
probes used for qMSP assay were as follows; for M-PDE9A,
forward primer: 59-CGG TGA GTG CGC GTC GC-39, reverse
primer: 59-CCA ACC ATC CCG AAA AAG CG-39, and probe:
59-TTC GGT GAC GTT ACG CGG T-39, for U-PDE9A,
forward primer: 59- GGT TTG TTT TGG TGA GTG TGT
GTC GT-39, reverse primer: 59- CCC AAC CAT CCC AAA
AAA GCA-39, and probe: 59-TTT GTT TGG TGA TGT TAT
GTG GTT T-39. The probes for M-PDE9A and U-PDE9A were
dual-labeled with 6-carboxyfluorescein (FAM) and minor groove-
binding non-fluorescent quencher (MGBNFQ). The thermal
profile consisted of an initial denaturation step of 95uC for 10
minutes followed by 50 cycles of 95uC for 15 seconds, 60uC for
30 seconds, and 72uC for 30 seconds. Calibration curves were
prepared for each assay by serial dilutions of single-stranded
synthetic DNA oligonucleotides specific to the M-PDE9A and U-
PDE9A amplicons. The sequences of the synthetic DNA were as
follows; for M-PDE9A: 59-CGG TGA GTG CGC GTT GCG
GGT TTT GTT CGG TGA CGT TAC GCG GTT TTT TCG
TTT TTT CGG GAT GGT TGG-39, for U-PDE9A: 59-GGT
TTG TTT TGG TGA GTG TGT GTT GTG GGT TTT GTT
TGG TGA TGT TAT GTG GTT TTT TTG TTT TTT TGG
GAT GGT TGG G-39.
Levels were calculated as copies/mL as previously described
[12]. Strict precautions were taken to prevent contamination,
and multiple negative-control water blanks were included in
every analysis. To reduce inter-experimental variation, each
extraction was repeated in triplicate. The final data reflect the
means of the results (Table S2 and S3). All samples were
analyzed blindly.
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Statistical Analysis
The clinical characteristics of the study population were
compared between cases and controls using the Mann-Whitney
U test and the x2test. The accuracy for detecting fetal DS was
analyzed with factors such as U-PDE9A level and unmethylation
index (UI). The UI is calculated as {U-PDE9A level/(M-PDE9A
level+U-PDE9A level)}X100. The accuracy of fetal DS detection
was determined with the fetal karyotyping results. Receiver
operating characteristics (ROC) curve analysis was performed to
assess the optimal cutoff value as described in our previous study
[13]. The optimal cutoff was set at a 5% false-positive rate for
comparisons with the accuracy of current screening test for fetal
DS. The sensitivity, positive predictive value (PPV), negative
predictive value (NPV), and odds ratio (OR) were calculated with
95% confidence intervals (CIs) using the EpiMax Table Calcu-
lator (http://www.healthstrategy.com/epiperl/epiperl.htm). Over-
all accuracy was estimated according to the area under the ROC
curve (AUC). Multiple logistic regression analysis was used to
estimate the values of U-PDE9A levels and UI as risk factors for fetal
DS, controlling for potential confounding factors. Potential
confounding factors included maternal age, body mass index, and
nulliparity at the time of blood sampling. Adjusted ORs (adjORs)
and their 95% CIswerecalculated. In all tests,thresholds of P,0.05
were set for statistical significance. Statistical analyses were
performed using the Statistical Package for Social Sciences 12.0
(SPSS Inc., Chicago, IL, USA).
Results
The clinical characteristics are given in Table 1. At blood
sampling, maternal age, gravidity, and body mass index did not
significantly differ between the cases and controls (P.0.05 for all).
The median gestational age was 7.5 weeks in both case and control
groups. Forty-four percent of participants in the case group and
41% of participants in the control group were nulliparous; this
difference between cases and controls was not significant
(P=0.799). Fetal gender-ratio and tobacco use also were not
different between the two groups (P.0.05 for both).
In the analysis of M-PDE9A and U-PDE9A levels according to
fetal gender, median levels of M-PDE9A were 1956.0 (interquartile
range: 1592.5–2411.2) and 2023.1 (interquartile range: 1650.0–
2310.3) copies/mL in male and female fetus-bearing participants,
Figure 1. Bisulfite genomic sequencing of PDE9A. The red characters in the sequences indicate methylation CpG sites of the PDE9A gene.
Methylated CpG sites of PDE9A were detected in maternal blood cells and placental tissues. Unmethylated CpG sites of PDE9A were detected only in
placental tissues. Yellow boxes indicate the regions that were amplified by the methylation-specific polymerase chain reaction.
doi:10.1371/journal.pone.0027709.g001
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respectively. Levels of U-PDE9A were 213.6 (interquartile range:
155.1–308.4) and 209.6 (interquartile range: 156.3–283.5) copies/
mL in male and female fetus-bearing participants, respectively.
Levels of M-PDE9A and U-PDE9A were not significantly different
between fetal genders (P.0.05 for all, Fig. 2). In the analysis of M-
PDE9A and U-PDE9A levels according to fetal karyotype, levels of
M-PDE9A were not different between the DS cases and controls
[1854.3 (interquartile range: 1653.3–2446.3) vs 2004.5 (interquar-
tile range: 1605.2–2316.9)copies/mL, P=0.928, Fig. 3]. However,
levels of U-PDE9A were significantly higher in the cases compared
to controls [356.8 (interquartile range: 314.8–437.4) vs 194.7
(interquartile range: 143.0–263.6) copies/mL, P,0.001, Fig. 3].
The accuracies of U-PDE9A level and UI for the detection of
fetal DS are shown in Table 2. The cutoff value for each factor was
set at the 5% false-positive rate by ROC analysis. Sensitivity, PPV,
and NPV were compared at an equivalent false-positive rate. The
ROC curves of U-PDE9A level and UI are presented in Fig. 4. The
U-PDE9A cutoff level of 320 copies/mL had a sensitivity of 77.8%,
PPV of 73.7%, and NPV of 95.5% in differentiating women
carrying a DS fetus from women carrying a normal fetus, and the
AUC was 0.907 (95% CI: 0.833–0.981) with a standard error (SE)
of 0.038 (P,0.001). The UI cutoff value of 14 had a sensitivity of
83.3%, PPV of 75.0%, NPV of 96.6%, and the AUC was 0.927
(95% CI: 0.851–1.003) with an SE of 0.039 (P,0.001). These
findings were confirmed using sample karyotypes (Table S1).
We analyzed the accuracy of U-PDE9A levels and UI for
detecting fetal DS based on gestational weeks at blood sampling
(Table 3). The sensitivity of U-PDE9A level was higher at 9–12
weeks of gestation than at 5–8 weeks of gestation, whereas the
sensitivity of UI was higher at 5–8 weeks of gestation than at 9–12
weeks of gestation. The specificities of both tests were higher at 9–
12 weeks of gestation than at 5–8 weeks of gestation. All cases
showing false-positive and false-negative results were observed at
or before a gestational age of 10 weeks (Table 4).
We analyzed the associations between each factor and the risk
of DS. As listed in Table 5, the risk of DS was significantly
increased in women with values greater than the cutoff values
compared with women whose values were less than the cutoff
values for U-PDE9A level or UI (OR [95% CI]: 59.5 [12.1–340.7]
for U-PDE9A level, 85.0 [15.5–564.2] for UI). After adjusting for
potential confounding factors such as maternal age, body mass
index, and nulliparity at blood sampling, the adjORs of U-PDE9A
level and UI were 46.2 (95% CI: 7.8–151.6) and 63.7 (95% CI:
23.2–206.7), respectively. Women with U-PDE9A levels and UI
greater than the cutoff values were at dramatically higher risk of
fetal DS in both unadjusted and adjusted analyses, compared with
women with values less than the cutoff values.
Discussion
Our findings demonstrate that U-PDE9A and UI are effective
biomarkers for the non-invasive detection of fetal DS during the
Table 1. Clinical characteristics in cases and controls.
CharacteristicsCase (N=18)Control (N=90)P value
At blood sampling
Maternal age (years)36.1 (31.3–38.0)31.1 (29.1–34.8) 0.059a
Gestational age (weeks)7.5 (6.0–9.0)7.5 (6.0–9.0)0.999a
Body mass index (kg/m2) 22.2 (20.6–24.0)21.4 (19.3–23.3) 0.180a
Nulliparity8 (44.4)37 (41.1)0.799b
Gravidity2.0 (1.0–3.3)2.0 (2.0–3.0)0.331a
Gender-ratio of fetus
(male:female)
7:1144:460.606b
Tobacco use 1 (5.6) 6 (6.7)0.999b
Values are median (interquartile range) or number (%).
a: Mann-Whitney U test, b: x2test.
doi:10.1371/journal.pone.0027709.t001
Figure 2. Levels of M-PDE9A and U-PDE9A in maternal plasma according to fetal gender. The upper and lower limits of the boxes and the
lines across the boxes indicate the 75th/25thpercentiles and the medians, respectively. The upper and lower error bars indicate the 90thand 10th
percentiles, respectively. The circles indicate outliers. Levels were analyzed by the Mann-Whitney U-test.
doi:10.1371/journal.pone.0027709.g002
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first trimester of pregnancy. We determined that U-PDE9A levels
and UI were significantly elevated in pregnant women carrying
DS fetuses compared to women carrying normal fetuses in the first
trimester, regardless of fetal gender. Moreover, pregnant women
with high values for U-PDE9A and UI were at an increased risk of
carrying a DS fetus. Therefore, we suggest that U-PDE9A and UI
may be useful biomarkers for non-invasive fetal DS detection using
circulating fetal DNA from maternal plasma, regardless of fetal
gender.
Current screening tests for fetal DS detection increases detection
rate by integration of multiple methods, such as measurement of
nuchal translucency and blood tests for numerous maternal serum
markers, including alpha fetoprotein, unconjugated estriol, human
chorionic gonadotrophin, and inhibin-A, but at a higher screen-
positive rate (approximately 16–22%) [3,4]. Moreover, diagnostic
test currently requires obtaining samples of fetal cells directly from
the uterus for genetic analysis, either through CVS between 11
and 14 weeks gestation or amniocentesis after 15 weeks. However,
these invasive procedures present a small but significant risk of
miscarriage in normal pregnancies [5,6]. Therefore, the develop-
ment of non-invasive prenatal tests for DS detection using
circulating fetal DNA in maternal plasma has been considered
as a potential alternative to improve current screening tests and to
reduce the need for invasive procedures, such as amniocentesis or
CVS.
Epigenetics is the study of molecular phenomena that affect
gene expression, but which do not involve alterations in DNA
sequences. The best-studied epigenetic phenomenon is the process
of DNA methylation, which suppresses gene expression and
Figure 3. Levels of M-PDE9A and U-PDE9A in maternal plasma according to fetal karyotype. The upper and lower limits of the boxes and
the lines across the boxes indicate the 75th/25thpercentiles and the medians, respectively. The upper and lower error bars indicate the 90thand 10th
percentiles, respectively. Levels were analyzed by the Mann-Whitney U-test.
doi:10.1371/journal.pone.0027709.g003
Figure 4. The ROC curves of U-PDE9A level and unmethylation
index. The ROC curves of unmethylation index and U-PDE9A level are
represented by blue and red lines, respectively.
doi:10.1371/journal.pone.0027709.g004
Table 2. The utility of measurements for detection of fetal
Down syndrome.
CutoffSensitivity PPVNPV
U-PDE9A level (copies/
mL)
320 0.778
(0.557–0.891)
0.737
(0.537–0.863)
0.955
(0.912–0.982)
Unmethylation index 140.833
(0.635–0.940)
0.750
(0.563–0.855)
0.966
(0.923–0.990)
The cutoff was set at 5% false positive rate by ROC curve analysis. To compare
detection accuracies of factors, sensitivity, positive predictive value (PPV), and
negative predictive value (NPV) were calculated at equivalent false positive rate.
The number in parentheses indicates the 95% confidence interval.
doi:10.1371/journal.pone.0027709.t002
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