NON INVASIVE PRENATAL DIAGNOSIS OF ANEUPLOIDY:
NEXT GENERATION SEQUENCING OR FETAL DNA
ENRICHMENT?
Webb A, Madgett TE, Miran T, Sillence K, Kaushik N, Kiernan M, Avent ND*
*Corresponding Author: Professor Neil D. Avent, School of Biomedical and Biological Sciences, Faculty of
Science and Technology, A411 Portland Square, Drake Circus, Plymouth, Devon, PL4 8AA, UK; Tel.: +44-
(0)1752-584884; Fax: +44-(0)1752-584605; E-mail: neil.avent@plymouth.ac.uk
page: 17
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INTRODUCTION
Current invasive diagnostic techniques pose
a risk to mother and fetus. The National Health
Service (NHS) offers prenatal screening to all pregnant
women in England [1]. At 11 to 13 weeks gestation
a combination of tests (termed the combined
test) are performed to screen for abnormalities and
score the risk of the fetus having Down’s syndrome
(trisomy 21) (T21). The combined test includes ultrasound
scans to check nuchal translucency and
analyzing maternal blood samples for free b human
chorionic gonadotrophin and pregnancy-associated
plasma protein A concentrations. These are combined
with factors such as maternal age, pregnancy
histories and familial genetic conditions [1]. Women
with high risk pregnancies are then offered prenatal
diagnosis. Current diagnostic procedures take samples
for karyotyping through invasive means, posing
a risk to mother and fetus.
Chorionic villus sampling (CVS) can be performed
from 10 weeks; taking a tissue sample
from the placenta through the cervix or abdomen.
Amniocentesis can be performed from 15 weeks by
obtaining a sample of amniotic fluid by passing a
needle through the mother’s abdomen into the uterus
[1]. The rate of miscarriage associated with CVS
and amniocentesis is 1.0 to 2.0%. Despite the risks,
on average 5.0-10.0% of pregnant women chose to
undergo these tests [2], averaging approximately
S 23,700 invasive diagnostic tests per year [1]. The
combined test has a 5.0-9.0% false positive rate
[3,4]; consequently mothers with healthy fetuses
may choose to undergo unnecessary invasive diagnostic
procedures, putting the fetus at risk of iatrogenic
spontaneous abortion. Non invasive prenatal
diagnosis (NIPD) would pose no such risk.
Increasing maternal age increases risk of Down’s
syndrome, Edwards’ syndrome (trisomy 18) (T18)
and Patau’s syndrome (trisomy 13) (T13), the three
most common autosomal aneuploidies in live births.
In the early 1970s, about 5.0% of pregnant women
were 35 years or over. However, almost 20.0% are
now over 35, increasing the incidence of aneuploid
fetuses [5]. Trisomy 18 and T13 are currently not
screened for until fetal anomaly ultrasound screening
at 18 to 20+6 weeks [1]. However, the NHS state
that although abortion is legal up to 24 weeks, it
should be carried out as early as possible, ideally
before 12 weeks [6]. Therefore, preferably, detection
of all aneuploidies would be made within the
first trimester, which would give greater scope for
parental choice. It is hoped that NIPD could help
achieve this.
Despite increasing maternal age, approximately
80.0% of Down’s syndrome patients are stillborn
to those under 35 [7]. Currently diagnostic tests
are only offered to those with high risk pregnancies
(such as those with abnormal serum protein levels)
as the risk of having a fetus with an abnormality
must be balanced against the risk of miscarriage.
This inevitably results in some abnormalities being
missed as women under 35 years are not considered
at risk of having a Down’s syndrome fetus [7]. Non
invasive prenatal diagnosis would allow testing
of these low-risk pregnancies. A recent European
Commission (EC) funded project, SAFE (Special
non-invasive advances in fetal and neonatal evaluation)
explored a number of new technologies in
NIPD [8,9], and this has been extended in a recent
project funded by the UK National Institute for
Health Research (NIHR), RAPID, and a EC framework
7 program, Eurogentest 2.In summary, prenatal testing is important as
it allows possible health issues of the baby to be
identified before birth, allowing arrangements for
immediate care to be made. It also enables parents
to make an informed choice regarding whether to
terminate the pregnancy. Replacing current invasive
tests with NIPD would reduce risk and increase detection
rate.
Methods for Non Invasive Prenatal Diagnosis
of Aneuploidies. The importance of prenatal diagnosis
and risk posed by invasive techniques makes
NIPD research a worthwhile commitment morally
and commercially. Research originally focused on
fetal nucleated cells found in maternal circulation
[10-12]. A number of cell types were investigated
but subsequently ruled out for a variety of reasons.
These include lack of fetal specific markers [13],
entrapment in the maternal lungs, confined placental
mosaicism [14] and persistence in the maternal
circulation years after pregnancy [15]. Furthermore,
only one or two fetal nucleated cells are found per
mL of maternal blood, further hampering their use
[7]. Research now focuses on cell free fetal DNA
(cffDNA), which was discovered in maternal plasma
in 1997 [10]. It has since been reported to be
present from as early as 4 weeks gestation [16],
making it possible for NIPD to be achievable earlier
in pregnancy than invasive methods. It has a
short circulation half-life (»16 min.) and is undetectable
in the maternal circulation within 2 hours
postpartum [17], making it specific to the current
pregnancy. Technical challenges stem from the fact
cffDNA constitutes only 3.0 to 6.0% of cell free
DNA in maternal plasma [7,10]. Detection of paternally
inherited alleles in maternal plasma is qualitative;
however, direct detection of aneuploidies is
reliant on dose, therefore quantitative. Therefore, it
was originally assumed that direct measurement of
fetal chromosome dose in maternal plasma would
show maternal chromosome dose and fetal chromosome
dose would be lost in the background of
maternal DNA. Nevertheless, chromosome dose
methods have been developed and are discussed
later. Methods for aneuploidy detection originally
focused on fetal markers for allelic ratio analysis.
Non Invasive Prenatal Diagnosis in Routine
Clinical Practice 2012. Following the discovery
of cffDNA in maternal plasma in 1997 [10], testing
for fetal RHD blood group status rapidly evolved
from risky DNA testing of amniotic fluid samples
provided from Liley curve investigations [18] to the
world’s first routine application of a non invasive
prenatal test clinically [19]. Fetal blood grouping
using maternal plasma as a source of fetal DNA is
now used extensively worldwide [20]. In addition, the detection of other paternally inherited alleles or
chromosome is possible, for example, fetal sexing
(for review, see [21]). Most clinically applicable
methods utilize the simple real time polymerase
chain reaction (ReTi-PCR) approach, however,
methods to detect the most commonly requested
prenatal diagnostic test, for aneuploidy, require
more sophisticated approaches. This is primarily
due to the fact that maternal DNA is an admix of fetal
(derived from the placenta) and maternal DNA.
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