2008 Barcelona - European Society of Human Genetics

Prenental diagnostics
P03.13
Inconsistent findings between QF-PCR and karyotype
analysis for the prenatal diagnosis of common trisomies in
amniocentesis
S. Y. Park 1 , D. J. Kim 1 , M. H. Lee 1 , B. Y. Lee 1 , J. Y. Park 1 , H. M. Ryu 2 , J. Y.
Han 2 ;
1 Laboratory of medical genetics, Medical research institution, Cheil general
hospital and women’s healthcare center, Kwandong university School of medicine,
Seoul, Republic of Korea, 2 Department of Obstetrics and Gynecology,
Cheil general hospital and women’s healthcare center, Kwandong university
School of medicine, Seoul, Republic of Korea.
We report an amniocentesis case of discrepant results between QF-
PCR and karyotype analysis in the prenatal diagnosis of trisomy18 .
The result of rapid QF-PCR analysis from uncultured amniotic fluid
cells indicated double autosomal aneuploidy for chromosome 18 and
21 . However, subsequent cytogenetic analysis from the in situ cultures
showed only trisomy 21 karyotype with no evidence of mosaicism . Follow-up
studies were performed on the repeated amniocentesis, fetal
cord blood and placental tissue after termination of the pregnancy .
QF-PCR and karyotype results showed trisomy 21 from all sampls .
For chromosome 18, both QF-PCR and karyotyping from placental
tissue revealed a normal disomic chromosome 18 . Cord blood and
repeated amniotic fluid cells indicated the aneuploidy of chromosome
18 in QF-PCR, but normal disomy 18 with no evidence of mosaicism in
karyotyping. We supposed that this inconsistent finding was the result
of duplication of the paternal chromosome 18 in mitosis after the loss
of the homologous maternal chromosome 18 at an early postzygotic
stage .
P03.14
QF-PcR on native chorionic villous for false positive and
negative detection is a useful tool in fetal karyotyping by direct
method
F. R. Grati, B. Grimi, B. Malvestiti, F. Dulcetti, G. Frascoli, S. De Toffol, A. M. Di
Meco, A. M. Ruggeri, R. Liuti, S. Milani, A. Trotta, F. Maggi, G. Simoni;
Reasearch and Development, Cytogenetics and Molecular Biology, TOMA
Laboratory, Busto arsizio, Italy.
The prenatal diagnosis(PD) on chorionic villi (CV) more reliable for the
identification of fetal karyotype combines the cytotrophoblast (direct
method, STC) and mesenchyme (long term culture, LTC) analyses .
However, when the sample available is poor (5-10mg) the diagnosis
is generally performed liking better for the direct method in order to
avoid the maternal cell contamination . This condition is associated
with a higher false negative risk . In our 7 years experience on 24’237
CV prenatal diagnosis, we recognised 18 cases of type V True Fetal
Mosaicism (TFM) that, if they have been analysed only with STC they
would have been subject to a false negative result . The abnormality
found in mesenchyme of 17/18 was an aneuploidy for chromosomes
13,18,21,X,Y . In 11 of them the abnormal cell line was present in a homogeneous
form, hence, possibly recognizable by QF-PCR . On these
basis we performed a study to calculate in our survey the false positive
and negative risk if PD would have been performed only with direct
method and to evaluate in 267 poor samples (5-10mg) if QF-PCR on a
minor fragment allows detection of false positive and negative cases .
We evidenced 1 false negative and 2 false positive cases and in 14
instances the abnormality found in cytotrophoblast was confirmed by
QF-PCR . In conclusion, QF-PCR could be an additional useful tool,
which doesn’t jeopardize the result of direct method, to decrease the
false negative risk from 1/1136 to 1/2941 .
P03.15
Rapid prenatal diagnosis of aneuploidies by QF-PcR: evaluation
of large scale clinical application
V. Cirigliano1,2 , G. Voglino3 , E. Ordoñez1,2 , A. Plaja4 , C. Fuster2 , M. Adinolfi5 ;
1 2 Dept. Genetica Molecular, General Lab, Barcelona, Spain, Departament
de Biologia Cel•lular, Universitat Autònoma de Barcelona, Barcelona, Spain,
3Molecular Genetics and Cytogenetics Lab. Promea-Day Surgery, Torino, Italy,
4 5 Dept. Genetica, General Lab, Barcelona, Spain, University College London,
London, United Kingdom.
Introduction: Recently it has been shown that rapid QF-PCR can detect
the great majority of chromosome abnormalities in prenatal samples
despite being deliberately targeted to chromosomes 21, 18, 13, X and
Y . Main advantages of the assay are low cost, speed and automation
allowing large scale application .
Methods: We developed a QF-PCR assay that has been applied on
38 .000 clinical samples . Most frequent indications were increased biochemical
risk (32%) and advanced maternal age (30%), 6% of these
cases were also associated with increased nuchal translucency; parental
anxiety and abnormal ultrasound were present in 22% and 7%
of samples respectively . Cytogenetic analysis was performed in all
cases and results compared .
Results: All 1278 non mosaic aneuploidies involving chromosomes 21,
18, 13 X and Y were detected with 100% sensitivity and specificity;
several cases of mosaicism and partial trisomies were also identified.
QF-PCR detected 95% of clinically significant abnormalities diagnosed
by cytogenetic analysis in samples referred for abnormal ultrasound .
Affected pregnancies could be terminated without further waiting for
completion of fetal karyotype
Conclusions: Large scale application of QF-PCR could reduce the
load of prenatal cytogenetics if all pregnancies are carefully monitored
by non invasive methods . In cases of negative QF-PCR results cytogenetic
analyses might only be performed for fetuses with abnormal
ultrasound; affected pregnancies identified by QF-PCR can be terminated
in a few hours from sampling . In countries where large scale
cytogenetics is hampered by its cost and lack of technical expertise
QF-PCR may be used as the only prenatal diagnostic test .
P03.16
Prenatal diagnosis of trisomy 21 by real-time PcR on fetal DNA
from amniotic fluid
T. Gunel 1 , K. Şahin 1 , I. Kalelioglu 2 , R. Has 2 , H. Ermis 2 , K. Aydinli 3 ;
1 Istanbul University, Faculty of Science, Department of Molecular Biology and
Genetics, Istanbul, Turkey, 2 Istanbul University, Faculty of Medicine, Istanbul,
Turkey, 3 Istanbul University, Cerrahpaşa Faculty of Medicine, Istanbul, Turkey.
Trisomy 21 is the most common congenital anomaly . A region called
Down Syndrome Critical Region (DSCR), extending from DNA marker
D21S55 on chromosome 21, is critical for Down syndrome fenotype
such as morphological features, hypotonia and mental retardation .
Recently, there have been major advances performing in the screning
and prenatal diagnosis of Down Syndrome . With the advent of realtime
PCR, it is now possible to measure the nucleic acid concentrations
with high accuracy . This study was undertaken to establish a rapid
prenatal diagnosis of trisomy 21 using real-time PCR of fetal DNA
from amniotic fluid. Fetal DNA’s from the amniotic fluid of a mother
expecting a baby with regular trisomy 21 and 9 mothers at high risk
for regular trisomy 21 were isolated . Real-time PCR technique was
used to measure the dosage of DSCR3gene. The amplification plots
of 40 cycles for each sample was determined by the real-time PCR using
fetal DNA’s from the amniotic fluid. C t (“cycle threshold”: the PCR
cycle number at which a significant increase in the components of the
reaction is first detected) values for each sample were obtained and
the average C t values were calculated . The DSCR3 and GAPDH gene
dosage were statistically determined and a comparison was made between
the results of the risk group and the one with regular trisomy 21 .
The real-time PCR results showed the DSCR3 gene dosage of one
of 9 samples from the risk group was higher than the one with regular
trisomy 21 .
P03.17
introducing new methods in prenatal diagnosis can do harm
more than good. the need for a national strategy
I. A. Dhaifalah, V. Curtisova, J. Santavy;
Palacky universtiy, Olomouc, Czech Republic.
Screening programs (SP) is indispensable part of health care systems
in many countries . During the past 50 years new SP are becoming
available. The innovation in this filed have been transformed into applicable
medical strategies and procedures with great benefits for
patients . Approval of a test obligates its use . Missing guidelines and
legitimate to identify centers and laboratories performing the SP have
lead to more harm than good in some places .
There is an increasing need for a single national body which provides
authoritative, objective decisions on whether, and under what circumstances
new SP or diagnostic tests should be made available . Country’s
health system care, culture, demography and resources should
be evaluated . Approval of a test should be accompanied by how the
test might most efficiently be provided and when outdated tests can