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 quantitation 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, time-consuming 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 pregnancies 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.