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

Concurrent Sessions
Trophoblast and peripheral blood cell samples were obtained from
women, undergoing pregnancy termination in the first trimester. Trophoblast
isolation was efficient and the cell populations showed high
purity for trophoblasts (85-94% positive cells), with low fibroblast contamination
(0-12%- positive cells).
Isolation and identification of proteins was performed by proteomics
technology. The results of 2-D gel analysis revealed many differences
in protein expression between the maternal blood cells and placentaderived
trophoblasts. The most obvious protein spots that appeared
in the placenta samples but were absent in the blood samples were
isolated, purified and identified.
The proteins identified were: cytokeratin 7, cytokeratin 8, keratin 19,
Zeta globin, A-gamma globin, G-gamma globin, and annexin IV.
Antibodies against cytokeratin 7 (an intracellular antigen) and annexin
(a cell-surface antigen) were tested in trophoblast cell lines (JAR and
BeWo) and Chorionic Villus (CVS) cultures. Annexin staining was positive
in the JAR cell line only whereas both lines as well as CVS cultures
were positive for cytokeratin 7. The remainder of the proteins detected
by proteomics are currently being screened and the most promising
markers will be applied in trophoblast isolation from maternal blood
samples, the ultimate goal being culture expansion and karyotype
analysis of the isolated cells.
C28. APEX microarrays for the non-invasive prenatal diagnosis
of beta-thalassaemia
T. E. Papasavva 1 , I. Kallikas 2 , S. Papacharalambous 2 , A. Kyrri 3 , L. Kythreotis 3 ,
H. Roomere 4 , M. Kleantous 1 ;
1 The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus, 2 Archibishop
Makarios III Hospital,, Nicosia, Cyprus, 3 Cyprus Thalassaemia Center, Nicosia,
Cyprus, 4 Asper Biotech Ltd, Tartu, Estonia.
The recent discovery of relatively abundant quantities of cell free fetal
DNA in maternal plasma and serum has opened up new possibilities
for the non-invasive prenatal diagnosis. In Cyprus, seven different
beta-thalassaemia mutations account for over 98% of all cases with
IVSI-110 representing the 81% of the total. Therefore, the development
of a non-invasive method for the Cyprus population is based on
the detection of paternally inherited Single Nucleotide Polymorphisms
(SNPs) as well as the direct detection of paternal beta-thal mutations.
One of the approaches that are being developed is the Arrayed Primer
Extension (APEX) method on the Genorama® QuattroImager . We
developed a DNA chip called “thalassochip” that contains 29 beta-thal
mutations and 10 SNPs linked to the beta-globin locus. More informative
SNPs are being added constantly.
The specificity and the sensitivity of the approach were tested using
genomic DNA. We were able to detect as low as 30pg/μl of genomic
DNA. Fetal DNA in maternal plasma is about 90pg/μl. Moreover, the
specificity of the approach was determined using genomic DNA. Following
that, the procedure was applied on maternal plasma DNA for
2 SNPs and we were able to detect the paternal allele of the fetus for
those SNPs.
Although the results are preliminary, the approach is very promising.
All the SNPs and mutations that are currently on the thalassochip need
to be standardized. Moreover, more informative SNPs need to be added
validated and standardized.
C29. Prenatal diagbosis in Denmark after the introduction of
nuchal translucency screening
S. Kjaergaard1 , J. M. Hahnemann1 , L. Skibsted2 , L. Neerup3 , L. Sperling4 , H.
Zingenberg5 , A. Kristiansen6 , K. Brondum-Nielsen1 ;
1 2 Kennedy Institute, Glostrup, Denmark, Roskilde Hospital, Roskilde, Denmark,
3 4 Gentofte Hospital, Gentofte, Denmark, Herlev Hospital, Herlev, Denmark,
5 6 Glostrup Hospital, Glostrup, Denmark, Naestved Hospital, Naestved, Denmark.
In 2004, the National board of Health in Denmark recommended new
guidelines for prenatal diagnosis. Instead of restricting the offer of prenatal
diagnosis mainly to pregnant women over the age of 35 years,
all pregnant women shall have the possibility to opt for 1. trimester
screening including nuchal translucency measurement in the fetus and
a maternal blood sample (doubletest), allowing a combined risk calculation
that the fetus has Down syndrome (or other trisomies). If the
risk is over 1:300, an invasive procedure is offered with chromosome
analysis of fetal cells from chorionic villus sample or amniocentesis.
We have surveyed the outcome of prenatal diagnosis in 3 counties in
22
Denmark after gradual implementation of the new guidelines during
2004, 2005 and 2006. The area covers approx 1.1 mill inhabitants,
1/5 of the total population. A comparison was also made with national
figures obtained from the Central Cytogenetic Registry. Main findings
were: An overall reduction in invasive procedures (from approx. 11%
of all pregnancies to approx. 5%), a doubled number of trisomy 21
fetuses diagnosed prenatally, and a reduction in the number of children
born with Down syndrome by approx 50%. The maternal history
of the children born with Down syndrome revealed: decline of offer of
screening, risk calculation above cut-off limit, too late appearance in
pregnancy. Sex chromosome abnormalities were apparently not detected
more frequently prenatally, but triplodi and possibly trisomy 18
were diagnosed more often than previously.
Selected cases with mosaicim and pseudomosaicim will be presented.
C30. Prenatal detection of partial chromosome imbalance by
Quantitative Fluorescent PCR (QF-PCR)
V. Cirigliano1,2 , G. Voglino3 , E. Ordoñez1,2 , A. Plaja1 , C. Fuster2 ;
1 2 General Lab, Barcelona, Spain, Universitat Autonoma de Barcelona, Bellaterra,
Barcelona, Spain, 3Promea Day Surgery, Torino, Italy.
The QF-PCR assay allows prenatal diagnoses of chromosome aneuploidies
in a few hours after sampling.
Several markers are needed on chromosomes X, Y, 21, 18 and 13
to minimize homozygous or uninformative samples. However, if microsatellites
are selected along the examined chromosomes, QF-PCR
may also allow detecting partial trisomies.
We developed a QF-PCR assay including up to 10 markers on the
sex chromosomes, 8 on chromosomes 21 and 18 and 6 markers on
chromosome 13.
In the course of its application on over 30.000 clinical cases the test
allowed detecting 100% aneuploidies without false positive results. On
the other end, it was also possible to detect 11 different partial aneuploidies
resulted from unbalanced translocations, duplications, insertions
and other structural rearrangements. Chromosome 18 was involved
in 4 cases, 6 fetuses had either X or Y derived extra sequences
and in two more samples partial trisomies 21 or 13 were also readily
detected as trisomic patterns for two or more STRs. In 6 cases QF-
PCR result was crucial to achieve the correct diagnosis.
Rapid prenatal diagnosis by QF-PCR has proven to be efficient and
reliable in detecting major numerical abnormalities. In this study we
demonstrate that an appropriate marker selection can also allow identification
of partial chromosome imbalance.
The main advantages of the assay are its low cost, speed and automation
allowing high throughput of samples. QF-PCR reaches the
purposes of relieving anxiety of most parents within 24 hours from
sampling or to accelerate therapeutical interventions in case of abnormal
result.
C31. Screening and maternal transmission instability of
intermediate-size and premutation FMR1 alleles in 24,446
mother-newborn pairs from the general population
F. Rousseau 1,2 , S. Lévesque 1 , C. Dombrowski 1 , M. Morel 1 , R. Réhel 1 , J. S.
Côté 1 , J. Bussières 1 , K. Morgan 3 ;
1 URGHM, Centre de recherche du Centre hospitalier universitaire de Québec,
Quebec, PQ, Canada, 2 CanGeneTest consortium & Université Laval, Québec,
PQ, Canada, 3 McGill University Hospital Centre, Montreal General Hospital
Research Center, Montréal, PQ, Canada.
Background: To study the instability of FMR1 gene triplet repeats in the
general population, we screened a large prospective sample of 24,446
anonymized mother-offspring pairs to determine transmission instability
of intermediate-size (40-58 triplets) and premutation-size FMR1
alleles (59-230 triplets) in the general population.
Methods: We used a mini-Southern blotting method to screen all mothers
for FMR1 alleles of 40 to 58 triplets (n=676) or premutation-size
(59+ triplets, n=22). We studied transmission of these alleles to their
offspring using triplet repeat PCR with validation of the mother-child
link using six highly polymorphic microsatellite markers.
Results: Out of a subsample of 343 transmissions of normal-size alleles
(