RAPID AND NON INVASIVE PRENATAL DIAGNOSIS
Madjunkova S1, Sukarova-Stefanovska E1, Kocheva S2,Maleva I1, Noveski P1,
Kiprijanovska S1, Stankova K3,Dimcev P4, Madjunkov M5, Plaseska-Karanfilska D1,*
*Corresponding Author: Professor Dr. Dijana Plaseska-Karanfilska, Research Centre for Genetic
Engineering and Biotechnology “Georgi D. Efremov,” Macedonian Academy of Sciences and Arts, Krste
Misirkov 2, Skopje 1000, Republic of Macedonia; Tel: +389(0)23235-410; Fax: +389 (0)2-3115-434;
E-mail: dijana@manu.edu.mk
page: 39
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INTRODUCTION
Prenatal screening and diagnosis are routinely
offered at antenatal care clinic visits, and are important
in decision making about the continuation
of pregnancies affected by genetic conditions for
which there are no cures, and prevention through
therapeutic abortion is a reasonable option. Prenatal
screening is offered to all pregnant women and
include fetal ultrasonography and maternal serum
biochemistry to select the pregnancies at-risk
for chromosomal abnormalities. However, these
methods have limited sensitivities (60.0-75.0%)
and specificities (false positive rate of 5.0%). Even
when used in combination and taking into account
maternal age, the identification rate of affected fetuses
does not exceed 90.0% [one]. Prenatal diagnosis
is usually preformed for detection of chromosomal
aneuploidies or monogenic diseases in “high
risk” pregnancies. Diagnostic testing currently
requires a sample of fetal cells obtained either by
chorionic villus sampling (CVS) between 10 and 14
weeks gestation or by amniocentesis after 15 weeks
of gestation. However, these invasive procedures
carry a risk of miscarriage of around 1.0% [2]. Prenatal Diagnosis of Chromosomal
Abnormalities. Chromosomal abnormalities (numerical
or structural) occur in 1 of 160 live births,
with extra copies of chromosomes 21, 18, and 13
accounting for the majority of numerical alterations
that are not related to sex chromosomes. The
prevalence of trisomies is highest in the first trimester
because of subsequent miscarriage and demise
of aneuploid conceptuses during pregnancy [3].
Conventional cytogenetic techniques (karyotyping)
are usually used to detect aneuploidies and large (5-
10 Mb) rearrangements in fetal cells (amniocytes,
trophoblasts), however, these are time-consuming
(2-3 weeks), subjective (small rearrangements) and
expensive. The development of molecular methods
for the rapid, targeted detection of aneuploidies of
chromosomes 13, 18, 21 and the sex chromosomes
by quantitative fluorescent polymerase chain reaction
(QF-PCR) [4,5] using fetal DNA, do not provide
a genome-wide screen for unexpected imbalances,
but are rapid (24-48 hours), accurate and
inexpensive. Multiplex ligation probe amplification
(MLPA) is a recent technique for relative quantita-tion of up to 40 to 45 nucleic acid targets. Several
MLPA commercial kits are used for prenatal detection
of common aneuploidies (chromosomes 13, 18,
21, X and Y), common microdeletion syndromes
and subtelomeric copy-number changes, identification
of marker chromosomes, and detection of
familial copy-number changes in single genes [6-
8]. The most powerful technique for genome wide
screening is array comparative genomic hybridization
(aCGH), which has the potential to combine the
speed of DNA analysis with a large capacity to scan
for subtle genomic abnormalities (approximately additional
10.0% of karyotying) respective to the resolution
of the used arrays [9-11], but is expensive, timeconsuming
and requires a high degree of expertise.
Non Invasive Prenatal Diagnosis. The discovery
of cell-free fetal DNA (cffDNA) in maternal
plasma in 1997 opened up new avenues for prenatal
diagnosis [12,13]. Fractional concentration of
fetal DNA is ~10.0%, coexists with a background
of maternal DNA and is present in maternal plasma
from approximately the 6th gestational week [14].
Techniques, such as real-time PCR (ReTi-PCR)
and digital PCR, provide sufficient sensitivity for
reliable non invasive assessment of this cffDNA
pool for paternally inherited traits such as sex and
RHD status, offering possibilities for non invasive
prenatal diagnosis of X-linked disorders (such as
Duchenne/Becker muscular dystrophy, Hemophilia
A, Hemophilia B, etc.) and RhD incompatibility,
respectively [15,16]. By detecting the presence
of fetal-specific paternally inherited mutant alleles
in maternal plasma, diagnosis of autosomal
dominant diseases transmitted by the father could
be made non invasively, whereas the absence of
such alleles could be used to exclude fetal inheritance
of autosomal recessive diseases [14,17-20].
Quantification of cffDNA, specific fetal and maternal
DNA and mRNA single nucleotide polymorphism
allelic ratios have been used to detect fetal
aneuploidies, however, the limitations of these techniques
affect the accuracy of the diagnosis [21-23].
Improvements were made after the discovery of the
unmethylated SEPINB5 gene that turned out to be
the first sex- and polymorphism-independent fetal
DNA marker found in maternal plasma [24-27].
The differential methylation of placenta and maternal
blood provides a rich source of markers for
non invasive prenatal diagnosis, however, further
research is needed to render the techniques widely
applicable. Implementing the new and robust next
generation sequencing techniques in detection of
the fetal aneuploidy made the detection for Down’s
syndrome to have 98.6-100.0% sensitivity and 96.8-
97.9% specificity [28,29].
Prenatal Diagnosis of Monogenic Diseases.
Monogenic diseases are the second most frequent
indication for prenatal diagnosis. The incidence of
these diseases, depending on the population, is up
to 2.0% newborns. Although there are some biochemical
tests and ultrasound findings to screen
and identify pregnancies at-risk for specific monogenetic
disorders, still the diagnosis is usually established
after the fetus is born in couples with no
familial history of the disease. In families at-risk for
monogenic disease, prenatal diagnosis is used to
determine fetal health and to provide adequate management
of the pregnancy and prenatal or perinatal
treatment. The new developments in prenatal testing
using cffDNA and their translation into clinical
practice are going to make a difference in selection
of pregnancies at-risk for monogenic disorders that
need invasive testing.
Prenatal Diagnosis at the Research Centre
for Genetic Engineering and Biotechnology
(RCGEB)“Georgi D. Efremov,” Skopje, Republic
of Macedonia. In the last 20 years, the researchers at
the RCGEB “Georgi D. Efremov” have performed
more than 80 prenatal diagnoses for different monogenic
diseases, such as hemoglobinopathies, cystic
fibrosis, Duchenne/Becker muscular dystrophy, spinal
muscular atrophy, hemophilia A, Lesch Nyhan
syndrome, Rett syndrome, phenylketonuria, galactosemia,
pseudohypoaldosteronism, etc. [30,31].
The prenatal diagnosis was performed on fetal DNA
by using standard molecular genetic techniques for
direct diagnosis of the disease or by using informative
polymorphic DNA markers for indirect diagnosis.
In 2001, the rapid prenatal detection of the most
common chromosomal aneuploidies (chromosomes
13, 18, 21, X and Y) by the multiplex QF-PCR
(mQF-PCR) method was introduced at the RCGEB
“Georgi D. Efremov” [32,33]. We have developed
a one-tube mQF-PCR assay for amplification of
22 highly polymorphic short tandem repeat (STR)
markers (at least four by analyzed chromosome)
(Table 1). Since then, more than 2200 prenatal diagnoses
of common aneuploidies in at-risk preg-nancies have been performed using the mQF-PCR
assay as a stand-alone test [34]. It was also used in
the prenatal cases of monogenic diseases to control
maternal contamination of the fetal material. The
prenatal diagnosis was performed on genomic DNA
isolated from fetal cells collected by amniocentesis
or CVS. Maternal blood samples were analyzed
in all blood contaminated amniotic samples and
in most chorionic villi samples. No discordant results
were obtained when cytogenetic analysis was
performed in addition to QF-PCR. Polymorphic
duplications involving STR markers D13S631,
D21S1441, D18S978 or D18S535 were detected in
seven fetuses; in all fetuses the duplications were
inherited from one of the parents. Using this method
we were also able to determine the parental origin
of the aneuploidy [35,36]. In our experience, the
QF-PCR method is an efficient, rapid and reliable
method for prenatal diagnosis of the most common
chromosome aneuploidies. In addition, it can provide
information about the origin of the aneuploidy
and maternal contamination of the fetal material.
In some “high risk” pregnancies with normal
QF-PCR results, we have used MLPA kits to analyze
subtelomeric regions and common microdeletion
syndromes. In addition to this, aCGH has been
employed in prenatal diagnosis of a few fetuses
with specific abnormal ultrasound findings.
We have also evaluated the specificity and
sensitivity of the real-time quantitative PCR method
for non invasive fetal sex determination using
cffDNA from maternal plasma. Our initial results
showed that this is a promising approach for fetal
gender determination in pregnancies at-risk for a fetus
with an X-linked disorder [37]. Our recent study
of non invasive determination of fetal RHD status,
using cffDNA from maternal plasma in RhD negative
pregnant women, showed 100.0% concordant
results with those obtained on fetal DNA from amniocytes
or CVS. This is a promising test that can be
used in clinical practice for targeted anti-RhD prophylaxis
and improvement of management of RHD
fetomaternal incompatibility. Using a multi copy
marker on Y chromosome (DYS14), we have increased
the sensitivity and specificity of the non invasive
fetal sex determination using cffDNA. This
method will be used in the future for non invasive
fetal sex determination in pregnancies at-risk for
X-linked disorders. Our further plans include translation
of the non invasive tests using cffDNA for
diagnosis of monogenic disorders and chromosomal
aneuploidies into clinical practice.
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