2008 Barcelona - European Society of Human Genetics

Prenental diagnostics
be withdrawn .
Czech Republic have an old tradition in the filed of prenatal screening
programs with the first amniocentesis at 1971. Women aged 40 and
than of 35 was the indications tell the late 80s . when the triple test have
been introduced . Regular and detailed registration started at 1985 . In
the year 1990 the number of invasive testes were 3 % for about 20%
detection rate for Down syndrome and 20 % other chromosomal abnormalities,
in 1998 and 2006 it was 10% for 60 % and 18% for 80%
respectively .
From this point of view a short description of the effect of implementing
the new SP on our center and that over the country will be presented .
P03.18
Proteomic analysis of amniotic fluid in pregnancies with Turner
and Klinefelter Syndrome fetuses
A. Kolialexi 1 , A. Anagnostopoulos 2 , G. Tsangaris 2 , K. Vougas 2 , N. Papantoniou
3 , V. Bagiokos 1 , A. Antsaklis 3 , A. Mavrou 1 ;
1 Medical Genetics, Athens University, Athens, Greece, 2 Proteomics Research
Unit, Centre of Basic Research II, Biomedical Research Foundation of the
Academy of Athens, Athens, Greece, 3 31st Department of Obstetrics & Gynecology,
Athens University School of Medicine, Athens, Greece.
Objectives: To determine the protein composition of amniotic fluid coming
from pregnancies with normal, Turner and Klinefelter syndrome
fetuses .
Material & Methods: Proteomic analysis was performed in stored amniotic
fluid samples of eighteen 2 nd trimester pregnancies. In five cases
routine cytogenetic analysis had shown that the fetus had Turner syndrome,
in four Klinefelter syndrome and in nine cases the fetal karyotype
was normal (5 females and 4 males) . Samples were analysed by
Two-Dimensional Gel Electrophoresis (2DE), coupled with Matrix-Assisted
Laser Desorption/ Ionization Time of Flight Mass Spectrometry
(MALDI-TOF-MS) . Selected proteins were further evaluated by Western
blotting .
Results: As compared to controls TRFE, LUM, RETBP and ApoA1
were significantly increased in Turner syndrome cases, whereas
KNG1, THBR and ApoA4 were decreased .
Four proteins (Apo A1, ZINCA2, LUM and A1AG) were found to be
up-regulated in samples obtained from pregnancies with Klinefelter
syndrome fetuses and three (RETBP, A1AT and VDBP) were down
regulated .
Conclusions: Different sets of proteins were differentially expressed in
the various sex chromosome abnormalities . Since these proteins are
likely to cross the placenta and be detected in maternal plasma, if the
specificity of our results is verified, they may be used as biomarkers for
the noninvasive prenatal diagnosis of sex chromosome aneuploidies .
P03.19
Noninvasive prental diagnosis of fetal trisomy
F. Crea 1 , W. M. Puszyk 1 , V. Cirigliano 2 , M. A. Hulten 3 , M. Vatish 3 , R. W. Old 1 ;
1 University of Warwick Department of Biological Sciences, Coventry, United
Kingdom, 2 General Lab, Genetica Molecular, Barcelona, Spain, 3 University of
Warwick Medical School, Coventry, United Kingdom.
The discovery of fetal DNA as a small component of cell-free DNA in
the maternal blood circulation has driven developments in non-invasive
prenatal diagnosis (NIPD) for the past decade . Interest has focused
upon NIPD of fetal trisomy 21, Down syndrome . We have explored
DNA regions on human chromosome 21 with the aim of identifying
DNA regions that are differentially methylated between adult leukocytes
and placenta . These two tissues represent a model system for
cell-free fetal and cell-free maternal DNA, respectively, in the blood
plasma of pregnant women . Among 46 DNA regions analysed in this
model system, a single differentially methylated region located in the
AIRE gene promoter, was identified. Further analysis of the methylation
of this DNA in the blood plasma of pregnant women indicated that
its placental epigenetic signature was not consistently maintained in
cell-free fetal DNA . The inconsistency exposes an apparent limitation
of the adult leukocyte/placenta model system as a means of discovering
epigenetic DNA biomarkers for use in the NIPD of fetal aneuploidies
. Our data indicate that more direct strategies are required, using
cell-fee plasma DNA .
P03.20
Optimization of blood collection, fetal DNA isolation,
concentration and analysis of fetal DNA present in maternal
blood
G. Minarik1,2 , B. Vlkova2 , T. Szemes1,2 , P. Celec1,3 ;
1Comenius University in Bratislava Faculty of Natural Sciences, Department of
Molecular Biology, Bratislava, Slovakia, 2Geneton s.r.o., Bratislava, Slovakia,
3Comenius University in Bratislava Faculty of Medicine, Institute of Pathological
Physiology, Bratislava, Slovakia.
Invasive methods currently used in prenatal genetic diagnostics represent
a not negligible risk for the health of both the subjected pregnant
woman and fetus . Not suprisingly, the detection and analysis of fetal
DNA in maternal plasma, representing a non-invasive alternative, attracted
much attention in the past few years . In our study, we focused
on comparing protocols for blood collection, isolation, concentration
and detection of fetal DNA present in maternal plasma of pregnant
women at various phases of pregnancy . We analyzed effects of addition
or omission of 4% formaldehyde solution during blood collection
and surveyed the implementation of DNA concentration after isolation
procedure using either ethanol precipitation or vacuum concentration.
We screened for presence of Y-chromosome specific sequences
employing three different methods . Two were based on qPCR (one
designed in our laboratory, one with commercial system specifically
used for identification and quantification of degraded human DNA sequences)
and one was based on PCR and fragment analysis of amelogenin
loci on automated genetic analyzer . We have successfully
prepared fetal DNA suitable for reliable qPCR and PCR analysis of Ychromosomal
sequences with all three tested detection systems . The
comparison of the collection, isolation, purification and concentration
approaches as well as detection methods can be observed in detail on
our poster presentation .
P03.21
Early first trimester detection of fetal cells and DNA in maternal
blood
E. Guetta 1 , L. Gutstein-Abo 1,2 , A. Tulchinsky 1,2 , M. Baum 3 , A. Hourvitz 3 , G.
Barkai 4,2 ;
1 Danek Gertner Institute of Human Genetics, Sheba Medical Center, Tel-
Hashomer, Israel, 2 Department of Human Molecular Genetics and Biochemistry,
Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel, 3 IVF Unit,
Department of Obstetrics and Gynecology, Sheba Medical Center, Tel-Hashomer,
Israel, 4 Department of Obstetrics and Gynecology, Sheba Medical Center,
Tel-Hashomer, Israel.
Detection of Y-chromosome sequences in fetal cell-free DNA or cells
from maternal blood are useful tools in non-invasive prenatal diagnosis
that have evolved into a fetal gender test feasible at earlier pregnancy
stages compared to ultra-sound screening .
The goal of this study was to determine the earliest possible time-point
for trophoblast and fetal cell-free DNA detection and determination of
fetal sex as a model for comprehensive prenatal diagnosis . Maternal
blood samples were collected from IVF patients at 2, 3 and 4 weeks
post-implantation . Fetal sex was determined in cells with FISH probes
following magnetic sorting with HLA-G, a trophoblast marker . Y-chromosome
SRY sequences were detected in cell-free plasma DNA with
real-time PCR . The results were compared with the newborn gender .
Fetal sex was accurately determined in cell-free DNA in 12/16 (85%)
of the samples, with 2 false-positive and 2 false-negative results . Trophoblast
testing yielded 8/15 (53%) accurate fetal gender determination
with 4 false-positive and 3 false-negative results . False-positive
results probably reflect the presence of a non-developing male embryo
as evidenced in one sample in which this explanation was supported
by PGD analysis .
The IVF samples yielded less accurate results compared with our ongoing
study of late 1 st trimester samples (weeks 7-13, parallel to 5-11
weeks post-implantation) in which 98% accuracy of fetal sex determination
was achieved with cell-free fetal DNA and trophoblast methods .
The uniqueness of the sample group - IVF patients - in which multiple
embryos are usually implanted, and the early stage of pregnancy, can
explain this discrepancy .