European Human Genetics Conference 2007 June 16 – 19, 2007 ...

Prenatal diagnosis
agreement with while direct short-term cultures karyotype), while the
long-term cultures showed a 45,X karyopype. Moreover one case was
partially informative because QF-PCR showed only one informative
polymorphic marker for trisomy 18. The karyotype of the long-term cultures
showed a trisomy 18. Our results confirm that QF-PCR technique
is a rapid testing able to diagnose chromosome aneuploidy accurately
in prenatal diagnosis on amniotic fluid but on chorionic villi more controversial
results can be obtained.
P0480. Detection of fetal aneuploidies by quantitative
fluorescent polymerase reaction
Z. Ban, B. Nagy, L. Lazar, G. Nagy, Z. Papp;
Semmelweis University, I. Dept. of Ob/Gyn., Budapest, Hungary.
Introduction: Multiplex quantitative fluorescent polymerase chain reaction
(QF-PCR) analysis of amniotic fluid samples has been shown to
be a useful tool in the detection of fetal aneuploidies, but has its limitations
as it can not detect some fetal chromosome disorders of clinical
importance.
Objective: To test the reliability of QF-PCR for the prenatal diagnosis
of the common aneuploidies, to obtain data on the allele distribution of
7 different short tandem repeats in Hungarian population and to analyze
the indications in which QF-PCR can be applied safely in prenatal
diagnosis.
Materials and methods: At 4985 patients (25 twin pregnancies) undergoing
amniocentesis we compared the results of QF-PCR with those
of the conventional cytogenetic study. We have analyzed allele distribution
of the applied STR markers. We compared the occurrence of
chromosome disorders which can not be detected by amnio-PCR in
cases with different indications of amniocentesis.
Results: 98.3% of amnio-PCR results were informative without falsenegative
and false-positive results. 9 samples (0.16%) were inconclusive
because of borderline peak ratios of diallelic results. 126
chromosomal abnormalities of 152 were detectable by amnio-PCR.
Amnio-PCR detected all chromosomal disorders in case of maternal
age as indication for amniocentesis. In case of structural abnormalities
detected by ultrasound, the chromosome disorder was not detectable
by amnio-PCR in 23 cases.
Conclusion: All chromosome disorders of clinical significance were
detected in case of advanced maternal age. The highest number of
chromosome disorders that are not detectable by QF-PCR was in case
of structural abnormalities detected by ultrasound.
P0481. Preparation of CVS for QF-PCR aneuploidy diagnosis and
correlation with karyotype analysis of cultured cells
K. Mann, A. Hills, C. Donaghue, C. Mackie Ogilvie;
Guy’s and St Thomas’ NHS Foundation Trust, London, United Kingdom.
Aneuploidy mosaicism has been reported to occur in up to 1% of chorionic
villus samples (CVS), usually due to differences between the
cytotrophoblast and mesenchyme cell lineages. Karyotype analysis of
cultured metaphases from the mesenchyme gives an accurate prenatal
diagnosis in the majority of cases. QF-PCR analysis of two separate
villus tips taken from different regions of the CVS has proven to
be an accurate predictor of fetal status regarding chromosomes 13, 18
and 21. Prior to June 2005 more than 3000 CVS were processed in
our centre with no completely discrepant QF-PCR/karyotype results.
However, in the following 6 months three such results were identified,
all due to sample mosaicism and QF-PCR analysis of isolated
cell populations. We therefore now test a small aliquot of dissociated
cells prepared from 10-15 mg of CVS for cell culture. A case study
has shown the mesenchyme to contribute between 40-50% of the
DNA in these samples. Since this change in CVS preparation protocol,
1738 CVS have been tested by QF-PCR for autosomal trisomy, and 4
cases of mosaicism detected; 60% trisomy 13, 78% trisomy 18, 30%
trisomy 21 and 35% trisomy 21. The first three cases showed nonmosaic
abnormal karyotypes with only the final case showing mosaicism
(46,XY,der(21;21)(q10;q10),+21[23]/46,XY[13]). In all cases the
mesenchyme cell lines were detected by QF-PCR and there were no
completely discrepant results. In summary, the QF-PCR analysis of
dissociated cells from whole CVS has resulted in better representation
of the CVS and greater concordance with the karyotype result.
1 1
P0482. DNA analysis with QF-PCR technique for confirmation of
suspected fetal triploidy
R. Raynova 1 , S. Andonova 1,2 , V. Dimitrova 3 , V. Georgieva 1 , I. Kremensky 1,2 ;
1 National Genetic Laboratory, University Hospital of Obstetrics and Gynecology,
Sofia, Bulgaria, 2 Molecular Medicine Center, Sofia Medical University, Sofia,
Bulgaria, 3 High-Risk Pregnancy Department, University Hospital of Obstetrics
and Gynecology, Sofia, Bulgaria.
We report three cases of fetal triploidy diagnosed by QF-PCR analysis.
The patients were referred to our Lab due to abnormal ultrasound
scan of both the placenta and fetus at 14 wg, 22 wg and 20 wg respectively.
QF-PCR technique was performed on DNA samples, extracted
with commercial kit from amniocytes (case 3) and from fetal
and placental tissue after termaniation of pregnancy (cases 1 and 2).
Fourteen polymorphic STR markers (4 located on chromosome 21, 4
- on chromosome 18, 3 - on chromosome 13 and 3 on chromosomes
X and Y) were amplified with Cy5-labeled primers. The observed electrophoretic
profiles for all investigated STR markers showed diallelic or
triallelic genotype concordant with trisomic pattern. No false negative
results were observed. For case 3 DNA samples from parents were
available and paternal origin of the additional chromosomal set was
proved. The triploidy was confirmed by cytogenetic analysis performed
after the termination of the pregnancy only in case 3. Our experience
showed that QF-PCR analysis is a reliable technique for rapid prenatal
diagnosis of triploidy when particular ultrasound anomalies are present
and karyotyping is not available. A high-standard ultrasound examination
was essential for the diagnosis of reported cases.
P0483. Evaluation of a non invasive prenatal RHD genotyping
screening strategy
M. Tsochandaridis 1 , R. Desbriere 2 , N. Lesavre 3 , C. D’Ercole 2 , J. Gabert 1 , A.
Levy-Mozziconacci 1,2 ;
1 Laboratoire de Biologie Moleculaire, CHU Nord, Marseille, France, 2 Service de
Gynecologie-Obstetrique, CHU Nord, Marseille, France, 3 Centre d’Investigation
Clinique, CHU Nord, Marseille, France.
It has been amply documented that fetal genotyping is feasible, allowing
NIPD for the identification of RhD positive fetuses in RhD negative
women. This approach now means that only those RhD negative women
who are known to carry an RhD positive child will require treatment
with anti-D IgG to prevent haemolytic disease of the newborn. We
described our experience with plasma obtained from 250 RhD negative
pregnant women between 10 and 28 weeks of gestation. 200 ul
of maternal plasma is automated extracted by biorobot EZ1 (Qiagen,
forencic card). The DNA eluate is tested in triplicate in RHD exon 7 and
10 real-time quantitative PCR. To reduce false negative due to low extracellular
nucleic acids concentration in plasma in some extractions,
we tested β-globin gene systematically. Plasmidic ranges for different
genes were also used. A strategy based on β-globin quantification
(cut-off at 1000 copies/ml) were established to perform this analysis in
routine. All RHD NIPD were compared with RH genotyping cord blood.
No false negative were obtained and two false positive cases were due
to the presence of RHD variants. In these two cases, using plasmidic
range permitted easily to confirm this diagnosis. In conclusion, automation
and quality control of extraction step are essential to introduced
this screening into restricted the antenatal anti-D immunoprophylaxys
to women carrying RhD-positive fetuses.
P0484. Prenatal findings in a severe type of achondroplasia
associated with mental retardation and acanthosis nigricans
(SADDAN).
J. A. Kortmann 1 , A. van Essen 1 , K. M. Sollie 2 , C. E. M. de Die-Smulders 3 , P. J.
Herbergs 3 , S. G. Robben 4 , M. M. Y. Lammens 5 , B. Timmer 6 , J. J. P. Gille 7 , M.
Vreeburg 3 , M. van Steensel 8 , I. Stolte-Dijkstra 1 , D. Marcus-Soekarman 3,9 ;
1 Department of Clinical Genetics, University Medical Center, Groningen, The
Netherlands, 2 Department of Gynecology and Obstetrics, University Medical
Center, Groningen, The Netherlands, 3 Department of Clinical Genetics,
Academic Hospital, Maastricht, The Netherlands, 4 Department of Radiology,
Academic Hospital, Maastricht, The Netherlands, 5 Department of Pathology,
University Medical Center St Radboud, Nijmegen, The Netherlands, 6 Department
of Pathology, University Medical Center, Groningen, The Netherlands,
7 Department of Clinical and Human Genetics, Free University Medical Center,
Amsterdam, The Netherlands, 8 Department of Dermatology, Academic Hospital,
Maastricht, The Netherlands, 9 GROW/University of Maastricht, Maastricht, The