2008 Barcelona - European Society of Human Genetics

Prenental diagnostics
P03.22
contamination-free analysis of single cells in cell-based noninvasive
prenatal diagnosis
T. Kroneis 1 , J. B. Geigl 2 , J. Waldispuehl-Geigl 2 , M. Alunni-Fabbroni 3 , E. Petek 2 ,
W. Walcher 4 , G. Dohr 1 , P. Sedlmayr 1 ;
1 Institute of Cell Biology, Histology and Embryology, Medical University, Graz,
Austria, 2 Institute of Human Genetics, Medical University, Graz, Austria, 3 Olympus
Life Science Research Europa, Munich, Germany, 4 University Clinic of
Obstetrics and Gynaecology, Medical University, Graz, Austria.
Analysis of very rare cells, as this is true for non-invasive prenatal
diagnosis (NIPD) based upon fetal cells circulating in the peripheral
blood of pregnant women present a formidable challenge in respect
of the need for non-ambiguous data . Currently used marker lack the
potential to exclusively discriminate fetal from maternal cells due to
unspecific staining, lack of fetal specificity, or low signal intensity. Thus,
pooling of fetal cells to enhance analysis efficiency in subsequent molecular
genetic analysis may give ambiguous data due to contamination
with maternal cells .
To circumvent maternal contamination while using the advantage of
pooling cells to increase analysis significance we are working on a procedure
to define a Post Identification Pool (PIP). The rationale is to use
markers with inherently limited specificity to (semi-automatically) detect
fetal candidate cells that are laser-microdissected and forwarded
to single cell whole genome amplification (scWGA). Aliquots of the latter
are then analysed by means of DNA fingerprinting using fluorescent
multiplex PCR. ScWGA products whose DNA profiles differ from the
maternal control sample are then pooled for further analysis .
Single fetal and maternal cells derived from one and the same interruption
material were subjected to PowerPlex 16 System® proofing
the DNA fingerprinting to be sufficient for discriminating fetal from maternal
cells. Currently a modified 16-fold multiplex is being set up to
perform DNA fingerprinting on pre-amplified (scWGA) single cells to
implement the PIP procedure .
P03.23
clinical practice of the incorporation of the non-invasive fetal
gender assesment in maternal blood
A. Bustamante Aragones, M. Rodriguez de Alba, I. Lorda-Sanchez, M. Trujillo-Tiebas,
M. Garcia-Hoyos, A. Avila-Fernandez, C. Ayuso, R. Carmen;
Fundacion Jimenez Diaz-Capio, CIBERER, Madrid, Spain.
Prenatal diagnosis is currently recommended to those pregnancies at
risk of an X-linked disorder however the invasive obstetric procedures
required entail a risk of miscarriage . Early fetal sex determination in
maternal blood can avoid the need for conventional PD in a half of
these cases . A previous large-scale validation study performed in our
laboratory concluded that this methodology was 100% accurate from
the 7 th week of gestation .
After incorporating this analysis into the clinical routine we have diagnosed
a total of 32 pregnancies at risk of an X-linked disorder including
cases of Haemophilia, Duchenne Muscular Dystrophy, Norrie
Disease . . . Two plasma samples were collected from each pregnant
woman in the first trimester of gestation: one from the 7 th week of gestation
and another from the 9 th week . Fetal gender was determined by
the presence/absence criteria of the SRY gene by Real-Time PCR .
Conventional prenatal diagnosis, ultrasound scan or gender at birth
confirmed that fetal sex was correctly diagnosed in maternal blood in
all cases . Early diagnosis of fetal sex in maternal blood represents a
great advantage for pregnancies at risk of an X-linked disease because
invasive prenatal diagnosis is suppressed in a half of the cases .
Since this diagnosis is performed before the 12 th week of gestation,
chorion villus sampling can be done in the case is required .
P03.24
Non invasive prenatal detection of two RHD gene exons and
fetal sex using cell free fetal DNA in maternal plasma
E. Ordoñez 1,2 , L. Rueda 1,2 , M. Lozano 1,3 , P. Cañadas 1 , C. Fuster 2 , V. Cirigliano
1,2 ;
1 General Lab, Barcelona, Spain, 2 Departament de Biologia Cel•lular, Universitat
Autònoma de Barcelona, Barcelona, Spain, 3 Institut Universitari Dexeus, Barcelona,
Spain.
Cell free fetal DNA (ffDNA) detection by Real-time PCR is routinely applied
for non-invasive genotyping of the fetal RhD status in reference
laboratories . The assay is easy to automate allowing high throughput .
We developed a new rtPCR for non-invasive prenatal RHD genotyping
and fetal sex determination using maternal plasma .
Two Taqman MGB-probes and primers were designed to develop a
Multiplex rtPCR for simultaneous amplification of exons 5 and 7 on
RHD gene . The multicopy DYS14 sequence on the Y chromosome
was also included in the assay . The test was evaluated blind on 50
coded plasma samples of known fetal genotype obtained from RhD
negative pregnant women, archived in our lab at -20Cº over the last
two years .
DYS14 products were detected in all 28 samples from male fetuses;
both RHD exons 5 and 7 were detected in 39 samples (23 males and
16 females) . No false positive were observed . Absence of all three
products indicating female RhD negative fetuses was observed in 6
cases . Fetal sexing results were 100% concordant, only in one sample
RhD exons failed to amplify resulting in an RhD negative female fetus
.
Even in old archived plasma samples multiplex rtPCR detection of
ffDNA was efficient and reliable allowing the assessment of fetal sex in
all cases . Only one sample from a RhD + female fetus was genotyped
as RhD - probably because of ffDNA degradation derived from repeated
thaw freezing cycles of the original plasma . The procedure proved to
be sensitive enough to be applied on clinical cases .
P03.25
simple and fast isolation of cell-free circulating DNA from
human plasma and serum
M. Meusel, C. Kirsch, G. Gutzke;
Macherey-Nagel, Dueren, Germany.
The discovery of cell-free circulating DNA in plasma opened up interesting
possibilities for noninvasive prenatal diagnosis as an alternative
to established invasive genetic screening procedures such as
amniocentesis and chorionic villus sampling . However, the isolation of
circulating DNA from plasma or serum is challenging . Circulating DNA
is highly fragmented and of very low concentration . Thus, established
nucleic acid purification protocols and ready-to-use kits have only a
very limited suitability for the extraction and purification of circulating
DNA . To overcome these limitations we developed the NucleoSpin
Plasma XS kit specially designed for the isolation of fragmented DNA
≥ 50 bp from human EDTA blood plasma. The kit exploits the benefits
of a unique binding column with a minimised dead volume and allows
for elution in 5-20 µl . Using up to 240 µl plasma the kit offers an easy
and convenient way for efficient purification of circulating DNA from
plasma . Data from kit development as well as application data, e .g .,
from fetal Rhesus D typing will be presented .
Besides prenatal genetic testing cell-free circulating DNA promises to
be applicable for the screening and assessment of a variety of pathological
findings such as cancer, stroke, myocardial infarction, inflammation
or trauma . The use of the NucleoSpin Plasma XS kit for these
applications will be exemplified.
P03.26
Update of EsHRE PGD consortium Activities
A. R. Thornhill;
The London Bridge Fertility, Gynaecology and Genetics Centre, London, United
Kingdom.
Since 1997, the European Society of Human Reproduction and Embryology
(ESHRE) Preimplantation Genetics Diagnosis (PGD) Consortium
has collected technical and outcome data, provided referral
networks, surveyed and promoted best practice . Membership increases
steadily (n=91) with increasing numbers of centres reporting (16-
45) and cycles reported (392-3358) between reports 1 and 7 . During
this time, reported cycle numbers for constitutional chromosome
abnormalities and monogenic disorders have increased with disproportionately
larger increases in preimplantation genetic screening
(PGS) cycles reflecting the increasing tendency for IVF laboratories
to select the ‘best’ embryo for transfer by elimination of chromosomally
abnormal embryos . Methodologies for every technical aspects of
PGD are becoming more sophisticated, accurate and reliable ensuring
extremely low misdiagnosis rates . PGD babies are comparable to
those derived from IVF with intracytoplasmic sperm injection procedures
with respect to pregnancy complications and congenital malformation
. The main complication, as with routine IVF, remains the risk
of multiple pregnancy and concomitant higher morbidity and mortality .