2009 Vienna - European Society of Human Genetics

Prenatal and perinatal genetics
P05.58
Partial submicroscopic duplications in markers D13s631 and
X22 detected with QF PcR
R. Raynova, S. Andonova, J. Genova, I. Kremensky;
National Genetics Laboratory, University Hospital of Obstetrics and Gynecology,
Sofia, Bulgaria.
Rapid diagnosis by Quantitative Fluorescent PCR analysis (QF-PCR)
has proved its cost-efficiency, speed and efficacy for prenatal detection
of the most common autosome aneuploidies - trisomy 21, trisomy
18, trisomy 13. For the past three years in our laboratory QF-PCR was
carried out on 2 300 prenatal samples (amniotic fluid, CVS). 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. Trisomy was indicated by
at least two informative markers on a single chromosome, showing
triallelic (1:1:1) or diallelic (1:2/2:1) trisomic pattern. In rare cases a
single marker result, consistent with trisomy may be observed, whilst
all other informative markers on the same chromosome are normal. A
partial submicroscopic duplication could be suspected. We have found
9 samples to have a single marker result consistent with trisomy - 8
samples showed trisomic pattern for marker D13S631, one for marker
X22. In cases when parental DNA was available we proved maternal
or paternal origin of the duplication. Thus we could distinguish the
rare inherited polymorphism from unbalanced translocation resulting
to partial trisomy.
P05.59
Large scale application of QF-PcR for rapid prenatal diagnosis
of common chromosome aneuploidies, results of nine years
clinical experience
V. Cirigliano1 , G. Voglino2 , E. Ordoñez1 , P. Cañadas1 , L. Rueda1 , C. Fuster3 , M.
Adinolfi4 ;
1 2 3 General Lab, Barcelona, Spain, Promea-Day Surgery, Turin, Italy, Universitat
Autònoma de Barcelona, Barcelona, Spain, 4University College London, London,
United Kingdom.
Despite being deliberately targeted to chromosomes 13, 18, 21, X and
Y the rapid QF-PCR test can detect the great majority of chromosome
abnormalities in prenatal diagnosis. The main advantages of the assay
are low cost, speed and automation allowing large scale application.
We developed a QF-PCR assay that was employed to test 43.000
clinical samples with results issued in 24 hours. The most common
referral indications were raised biochemical risk (32%) and advanced
maternal age (30%). All samples were also tested by conventional cytogenetic
analysis and the results compared.
A total of 1550 non mosaic aneuploidies involving chromosomes 21,
18, 13 X and Y were detected with 100% specificity. Several cases
of partial trisomies and mosaicism were also identified. Overall 95%
of clinically relevant abnormalities were readily detected and termination
of the affected pregnancies could be performed without waiting for
results of the cytogenetic analyses.Our results support the possibility
of reducing the load of prenatal cytogenetic tests if pregnancies are
carefully monitored by non invasive screening. As a result, invasive
procedures should only be performed in high risk pregnancies. In case
of abnormal QF-PCR results, medical action can be made available
in a few hours after sampling. In cases of negative QF-PCR results
cytogenetic analyses might only be performed for fetuses with clear
evidence of abnormalities based onl ultrasound results. In Countries,
where large scale conventional cytogenetic tests are hampered by
high cost and lack of technical expertise, QF-PCR may be used as the
only prenatal diagnostic test
P05.60
Rapid prenatal diagnosis by QF-PcR analysis on 700 cases
A. M. Stan1 , C. Dragomir1 , E. Severin2 , L. Savu1 ;
1 2 Genetic Lab S.R.L., Bucharest, Romania, Carol Davila University of Medicine
and Pharmacy, Bucharest, Romania.
The quantitative fluorescence PCR (QF-PCR) assay was recently introduced
in Romania in the prenatal diagnosis field allowing the rapid
detection of most frequent chromosomal aneuploidies involving chromosome
21, 18, 13, X and Y in high-risk pregnancies.
The main objective of this study was to demonstrate that QF-PCR
based assay is an efficient, reliable and rapid method that can reduce
the number of conventional cytogenetic analysis in carefully monitored
pregnancies. Therefore we performed rapid prenatal testing for 700
cases proceeding from small amounts of fetal sample including amniotic
fluid, chorionic villi and products of conception.
The methodology included the relative quantification of microsatellite
alleles to determine sequence copy number, using a multiplex QF-PCR
reaction and capillary electrophoresis. The multiplex QF-PCR reaction
was performed with a commercial available kit for rapid prenatal diagnosis
of trisomy 21, 18, 13 and sex chromosome aneuploidies.
We analyzed 675 amniotic fluid samples, 13 products of conception
and 12 chorionic villi samples. All samples with trisomy 21 (n=17), trisomy
18 (n=3), trisomy 13 (n=1), trisomy XXY (n=2), monosomy X
(n=2), triploidy (n=2) and mosaicism (n=2) were accurately diagnosed
without false-negative results. We detected somatic microsatellite mutation
in two cases and submicroscopic polymorphic duplications in
four samples, all six cases involving a single STR marker. All six cases
were reported as normal by cytogenetic analysis.
In conclusion we are confident that the QF-PCR approach is an efficient,
reliable and rapid prenatal diagnosis method offering an alternative
to conventional cytogenetic testing.
P05.61
Analysis of heterozygosity level of stR markers by QF-PcR
method for prenatal diagnostics in Northwest region of Russia
I. Belotserkovsky, G. Demin, T. Ivashchenko, I. Fedorova, V. Baranov;
Ott’s Institute of Obstetrics and Gynecology RAMS, Saint-Petersburg, Russian
Federation.
Last years prenatal diagnosis for the most common chromosome abnormalities,
such as Down’s, Edward’s, Patau’s syndromes and also
numerical sex chromosomes abnormalities has carried out by quantitative
fluorescent PCR (QF-PCR). QF-PCR is highly sensitive and specific
test revealing these chromosome abnormalities which account for
around 95% of all chromosome pathologies of the fetus. We have used
11 chromosome-specific of markers (DXS6854, D13S628, D13S634,
D13S742, D18S386, D18S380, D18S391, D18S535, D21S1270,
D21S1411, D21S226) to determine the heterozygosity level for 203
DNA samples from Northwest region of Russia individuals. The lowest
heterozygosity level was registered for D18S380 marker (59%) whereas
the highest level was found 89% for D13S742 marker. The mean
level of heterozygosity was found as 74%. When using two markers
on the same chromosome heterozygosity level was increased: 99%
- for chromosome 13; 91% - for chromosome 18 and 97% - for chromosome
21. Use of three and more markers on each chromosome
increases level of heterozygosity and informativeness of the method
up to 99-100%. Heterozygosity levels of D18S535 and D13S742 markers
were significantly different from English population. We found that
D13S742, D18S386 and D21S1411 markers are the most informative
for prenatal diagnostics in Northwest region of Russia.
P05.62
Quantitative real-time PcR technique for rapid and prenatal
diagnosis of trisomy 21 syndrome
A. R. Kamyab 1,2 , N. Masroori 2 , F. Maryami 1 , R. Mirfakhraie 3 , F. Maryami 1 , M.
Karimipoor 1 , R. Mahdian 1 ;
1 Department of molecular medicine, Pasteur institute of Iran, Tehran, Islamic
Republic of Iran, 2 Department of biology, science and research branch, Islamic
Azad university, Tehran, Islamic Republic of Iran, 3 Department of medical
genetics, National institute of genetic engineering and biotechnology, Tehran,
Islamic Republic of Iran.
Down’s syndrome (DS) is a genetic disorder that affects 1 in 700 live
births across all ethnic groups. Trisomy 21 syndrome is caused by
an extra copy of chromosome 21. Currently cytogenetic methods as
karypotyping and FISH are used for diagnosis of DS. However these
are labor and too time-consuming. On the contrary, molecular methods
such as Real-Time PCR are sensitive and high throughput. According
to the study, peripheral blood was collected from patient and
normal controls. Subsequently by salting out method, genomic DNA
was purified. In this study, two genes as DSCAM and DYRK1A2 (target
genes) and PMP22 (reference gene) locating on chromosome 21
and 17 respectively, selected and measured copy number of them in
trisomy 21 patients and normal individuals. Then, DSCAM/PMP22 and
DYRK1A2/PMP22 ratio was calculated by 2 -ΔΔCt formula. The results
of Real-Time PCR showed the ratio of 1.71±0.16 and 1.01±0.10 (p