|Numerical aneuploidy; Prenatal diagnosis; QF-PCR;
|Aneuploidy is the most common chromosomal aberration with
clinical importance in humans. It is of high frequency in embryos and
exists in 3 to 4% of recognized pregnancies and 1 in 160 live births
. Probability of aneuploidy occurrence increases with maternal age.
Aberrations of chromosomes X, Y, 13, 18 and 21 comprise about 95%
of all chromosomal disorders in newborns. Meanwhile, trisomies of
chromosomes 13, 18 and 21 are more important and trisomy 21 is the
most common . Screening for genetic diseases is a well-accepted
procedure as a preventive solution in many countries. Biochemical
analysis is the primary stage of any aneuploidy screening in pregnancies.
Ultrasonography is usually used in conjunction with biochemical tests
to find high risk pregnancies. The accuracy of these two methods has
been reported to be between 79-90% .
|Though these two methods are non-invasive, due to low accuracy,
alternative methods are used to increase the detection rate [4,5]. The
most widely used method is fetal karyotyping. Cytogenetic analysis
usually requires 15 to 25 days, a time of extreme anxiety for the couple
and their family. In many cases, late therapeutic abortions may be
risky. Furthermore, karyotype preparation and analysis are expensive
and difficult [6-8]. Therefore Rapid Aneuploidy Diagnosis (RAD) has
become a necessity. One such method is the use of FISH (Fluorescence In
Situ Hybridization). FISH is a molecular method in which chromosome
specific probes are labeled with fluorescent materials and then are
hybridized to chromosome spreads laid on slides [9-11]. The targets
are specific sequences of DNA, to which probe will bind. In aneuploidy
FISH, probes are specifically designed for the chromosomes X, Y, 13,
18 and 21 . FISH, though capable of diagnosing the chromosomal
aberration using the nucleated cells, is expensive, tedious, requires
dedicated probes and is not so easy to get good results in somewhat
inexperienced hand. Another rapid molecular test is the use of STR
(short tandem repeat) based quantitative fluorescent PCR (QF-PCR)
. QF-PCR as its stands for is a method in which by means of
fluorescent based PCR one can quantitate specific DNA copy number or DNA type, but is more used for detecting chromosomal aneuploidy
in prenatal testing.
|Publication of a number of papers regarding evaluation of the
STRs for the gene mapping proved their diagnostic application value
for identifying a group of aneuploidies [13,14]. Nowadays QF-PCR is
preferred over FISH. Markers used in aneuploidy QF-PCR are located
in regions of tested chromosomes with polymorphic alleles . The
microsatellites used in this method are 3-5 base pairs long .
|Though cytogenetic method is still the golden choice, rapid
methods are preferable since the risk of abortion-related mortality
increases with gestational age . Nowadays, QF-PCR is used in
prenatal diagnosis centers in Europe for diagnosing the most important
chromosomal numerical aberrations [15,18-24]. In countries like the
UK, new screening programs for Down Syndrome (DS) need not
include karyotyping and can offer diagnosis with RAD as a standalone
approach [20,25]. QF-PCR is the most preferable and easy to use
method as a RAD method.
|In the present study, aneuploidies were studied in 100 chorionic
villus (CV) samples, amniotic fluid (AF) samples and blood samples
collected from the pregnant women and patients and the results
obtained were compared with cytogenetic analysis results.
|Materials and Methods
|The samples were supplied from several Welfare Centers and medical
genetics laboratories. The samples included blood and fetal samples
(CV and AF). All samples were used after signing of informed consent
forms. The criteria for taking samples for prenatal diagnosis included
advanced maternal age (n=11), abnormal fetal ultrasonographic
signs with or without advanced maternal age (n=9), positive results
following maternal blood biochemical screening (n=25), previous
record of chromosomal aberration (n=9) and abnormal karyotype of
parents (n=1). The blood samples (n=45) belonging to patients who
had been referred to the medical genetics centers for confirmation of
DS or other chromosomal anomalies or placed in the national Welfare
Centers. Blood samples, of about 2 ml, were taken from the affected
persons and were spotted onto DNA Banking Cards (DBC) (Kawsar
Biotech Companies, KBC, Tehran, Iran). To extract the DNA, a 1.5
mm diameter circles were cut using a Micropunch (KBC, Tehran, Iran)
and rinsed using DBC extraction buffer (KBC, Tehran, Iran) for 2-3
times. The discs were used directly after the final rinse. DNA from
chorionic villi (CV) were extracted using trypsin and using method
described . DNA from amniotic fluid (AF) samples, were extracted
using the method described elsewhere . QF-PCR was done using
Aneufast kit as recommended (Genomed AG, Switzerland) (Table 1).
For all cases, initially S1 and S2 were used and if any confirmation was
needed chromosome specific kits (i.e. MXY, M12, M18, M13 for X and
Y, 13, 18 and 21 chromosomes respectively) were used according to
|For performing QF-PCR either one disc from the DBC or 5
microliter (μl) DNA was added to 10 μl of Aneufast multiplex mix.
PCR was done as an initial denaturation for 15 minutes, amplification
using 28 cycles at 95°C for 40 sec, 58°C for 80 sec, and 72°C for 40 sec,
and final extension for 30 sec, at 60°C. About 1.5 μl of each of the PCR
products together with 0.3 μl of the size standard of GeneScan™-500
LIZ™ was added to 40 μl of Hi-Di Formamide. The samples were
denatured for 2 minutes at 95°C and loaded into ABI 3130 XL Genetic
Analyzer (ABI, US). Cytogenetics was done as described . Analysis
was done using automated cytogenetics platform CytoVision and its
Software (Wetzlar, Germany).
|In this study, 100 samples including 45 blood, 43 AF and 12 CV
samples were analyzed. The DNA extractions were successful in all
the blood and CV samples, but it was unsuccessful in two of the AF
samples due to high contamination by maternal blood. These two gave
poor and unanalyzable results but subsequent purification using KBC
DNA cleanup kit (KBC, Tehran, Iran) and repeating the QF-PCR gave
good results. In general, the DNA extraction from the chorionic villus
samples was much simpler and more accurate than the AF, since the
maternal blood and blood clots can easily be removed prior to DNA
extraction from the CVs. Using the QF-PCR method the analysis results
of 90% of the samples were available within 48 hours but 10% of the
samples were re-analyzed because of uncertainty. Cytogenetic analysis
was performed within two-four weeks. Results of cytogenetics and
QF-PCR for each sample were analyzed independently and they were
compared after the end of the study. Several numerical chromosome
abnormalities were observed and are summarized in Table 2.
|Diagnosing normal samples
|Presence of at least two different STR peak for each chromosome
with a 1:1 ratio proves normality. All the blood samples in this analysis
had been taken from the individuals with chromosome aneuploidy;
so there were no normal cases. 10 samples (83.3%) out of 12 CV and
16 samples (37.2%) out of 43 AF were normal. Fetal sexing and the
numbers of sex chromosomes were determined through amplification
of the non-polymorphic sequences of amelogenin gene. This region
give two differing bands one for the Y and the other one for the X
chromosome. When both bands are present then the sample is regarded male gender otherwise the gender is regarded female. Other STR
markers confirm the above results and may show other forms of sex
chromosome abnormality like XXY which will not be detected using
the amelogenin gene. Detection of fetal sex was correctly performed
in all the blood samples, and no difference was observed between the
cytogenetic and the QF-PCR results.
|Autosomal AD was possible by the presence of at least three different
STRs as triallelic peaks (three alleles equal in size) or in diallelic trisomy
state (two alleles, one twice the size of the other) for each chromosome
proving it being a trisomy. 41 samples (91.1%) out of 45 blood samples
were recognized as trisomy 21. 16 samples (37.2%) out of 43 amniotic
fluid samples had trisomy 21 and three cases (6.98%) had trisomy 18.
None had trisomy 13.
|Sex chromosome AD
|Sex chromosomes were correctly recognized in all samples. One
case (2.22%) of Turner syndrome (XO) (X monosomy) and two cases
(4.44%) of Klinefelter (XXY syndrome) were recognized in blood
samples. In addition, one case (2.32%) of Turner syndrome, one case
of the Klinefelter syndrome and one cases of X trisomy (XXX) were
observed in amniotic fluid samples.
|In two AF samples, the QF result was different from that of
cytogenetics. One sample was diagnosed as trisomy 21, while the
cytogenetic analysis result recognized it as normal. This could be due to
mosaicism and preferential growth of normal cells during AF culturing
as seen before .
|The other sample was poorly identified as triploidy or tetraploidy
by QF method, while cytogenetic showed that the fetus had trisomy 21.
Maternal blood contamination may be the caused for controversial QF
result. DNA extracted from the AF cultured cells confirmed cytogenetic
|One sample detected to have mosaic triploidy by QF-PCR which
failed to provide cytogenetics result due to inability of the cells to grow.
There may other causes as well but this is more likely .
|In normal individuals who are heterozygous from STRs, an equal
amount of fluorescent is created for both alleles; so the proportion
between the areas and heights of the peaks will be almost 1:1. In normal
individuals, whose STRs' alleles are homozygous, would have the same
repeat number and size; so quantification would be impossible, and the
marker is not useful for the analysis. Nevertheless the markers have
been chosen as such that it is very rare to have a normal person to be
homozygote for all markers in the general kit and chromosome specific
kit. In a trisomy sample, three versions of a chromosome are diagnosed;
consequently, the chromosome STRs usually show three peaks with
almost identical fluorescent intensity and proportion of about 1:1:1
between the areas (triallelic trisomy) (Figure 1). In a trisomic situation
when two chromosomes have similar repeat unit (equal PCR product
size) and one a different size, the quantitative PCR produces two
imbalanced peaks with an area proportion of 2:1 (diallelic trisomy)
(e.g. D21S1435 marker in Figure 1).
|QF-PCR is a simple, rapid and cost effective method for prenatal
diagnosis of common chromosomal aneuploidies (e.g. 21, 18, 13, X and
Y). The result can be ready within 24-48 hours after sampling while
cytogenetics usually requires cell culturing and it usually takes more
than 14 days to produce enough cells to make cytogenetic preparation
possible. When human labor and other costs are compared for both
methods then the cost for the QF is almost half of the cytogenetics .
Another advantage of QF-PCR is the reduction of waiting time for
results by the families. Usually families are under immense stress after
giving the fetal sample and waiting for results (personal observation).
|One may argue that cytogenetic is more accurate therefore the
accuracy should not be compromised over speed or cost. This point has
been extensively reviewed and two major policies are under practice
. In the UK, the use of RAD methods are sufficient for aneuploidy
testing during pregnancies when only aneuploidy screening is the
aim. Nevertheless, in the USA "a joint statement by the American
College of Medical Genetics and the American Society for Human
Genetics reaffirmed that all RAD test results must be followed up with
karyotyping" [30,31]. QF-PCR is regarded the most cost effective and
preferable method for AD.
|In our study the QF-PCR diagnosed 100% of the trisomies 13,
18 and 21 and sex chromosome aneuploidies without false-negative
results, and the fetal detection was perfectly done in all the samples
(Table 2). It however, could not easily detect mosaicism (Table 2). This
is a particular problem when one type of karyotype is less represented
(e.g. less than 1:4-6 ratios). The STR markers in the Aneufast kit were
100% informative for all the samples. In at least one sample maternal
blood contamination gave unacceptable QF-PCR result. More care has to be employed during AF sampling to reduce maternal blood
contamination of the AF. When maternal blood is obscuring the
results, parallel testing of maternal DNA may resolve the problem or
one has to rely on cytogenetic results. Our strategy in this study was
that we would culture all AF samples and also perform the QF-PCR. If
QF was successful and clear result was obtained then we would inform
the family about the result but wait for the cytogenetic result to see if it
would confirm the QF-PCR outcome. This strategy would be violated
(i.e. relying only on the QF-PCR) if the gestational age is as such that
the cytogenetic results would become ready after the legal time limit for
therapeutic abortion (end of 18th weeks in Iran).
|Regarding limitations of diagnosing chromosomal aberration,
FISH method is similar to the QF-PCR method, while the QFPCR
has several advantages over the FISH method. Misdiagnosis in
contaminated samples is less in QF-PCR than FISH . Costs and
complexity of FISH are more than QF-PCR. The karyotype methods
also enjoy high accuracy, while being time consuming due to the need
for cell culture. Numerous studies have been performed to determine
the accuracy of chromosomal disorder diagnosis. Cirigliano et al.
performed an investigation on a large number of the samples during
a nine year period . Based on these studies, the QF-PCR method
was found to be capable of diagnosing the chromosomal aneuploidies
of chromosomes X, Y, 13, 18 and 21 with 100% accuracy; also above
95% of the chromosomal aberrations were diagnosed using the clinical
symptoms with ease and termination of pregnancy could be performed
without waiting for cytogenetic analysis results . In addition, in
a report recently published a sum up of the study results of several
scientists on QF-PCR method over a 15-year period has been given
[9,34]. The group announced that the pregnant women can perform
this analysis as an alternative to the cytogenetic methods. The major
disadvantage of this method is its inability to detect mosaic and
numerical aneuploidy of the chromosomes other than X, Y, 13, 18
and 21, as well as its inability to detect structural aberrations [9,34].
However, other chromosomal abnormalities (CA) are rare among
women who are under general screening program (with no prior
history or complication before screening). QF method is also capable
of detecting the origin of extra chromosome (Figure 2).
|Hereby, we would like to thank and appreciate esteemed colleagues at
Kariminejad & Najmabadi Pathology and Genetics Center, Akbari Medical Genetics
and PND Laboratory, the Tehran Welfare Centre and all esteemed friends and
our staff of Zeinali's Medical Lab., Kawsar Human Genetics Research Center who
have helped us in this study.
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