2008 Barcelona - European Society of Human Genetics

Prenental diagnostics
P03.51
mLPA validation for prenatal diagnosis of chromosomal
anomalies: retrospective study of 125 consecutive chorion villi
samples
D. Diego-Alvarez, C. Ramos, B. Gomez-Dominguez, R. Cardero-Merlo, A.
Bustamante-Aragones, M. Trujillo-Tiebas, C. Auz-Alexandre, J. Gallego-Merlo,
A. Gimenez-Pardo, F. Infantes, C. Ayuso, J. Diaz-Recasens, M. Garcia-Hoyos,
R. Riveiro-Alvarez, E. Vallespin, A. Avila-Fernandez, D. Cantalapiedra, M. Rodriguez
de Alba, I. Lorda-Sanchez;
Fundación Jiménez Díaz, Madrid, Spain.
Rapid screening for common aneuploidies (trisomy 13, 18, 21 and
those involving sexual chromosomes) by interphase nuclei FISH or
multiplex QF-PCR has become a common offer in prenatal diagnosis
. Although it reduces maternal anxiety by discarding the most frequent
chromosomal anomalies, some other like terminal deletions are
missed . In addition, rapid detection of unbalanced products of parental
balanced translocations requires the use of additional specific FISH
probes . In contrast, MLPA technique with subtelomeric probe-mixes
provides information about copy number changes of every chromosome
in a rapid (24 h) and simple protocol requiring few material . Although
specific probe-mixes for the detection of common anomalies in
prenatal diagnosis have been validated, the aim of the present study
was to evaluate the use of subtelomeric probe-mixes for the rapid detection
of aneuploidy, unbalanced reciprocal translocations and terminal
chromosomal rearrangements of every chromosome on a hundred
and twenty-five consecutive chorion villi samples . Terminal deletion or
duplication was suspected when two different probes for a chromosomal
arm appeared deleted or duplicated, respectively . Aneuploidy
was suspected if probes for both arms of a chromosome appear deleted
(monosomy) or duplicated (trisomy) . Results demonstrate the
capability of MLPA on detecting not only numerical but also structural
chromosomal anomalies on prenatal samples . Thus, this technique
can also be a rapid and simple approach for the detection of balanced
or unbalanced products of translocations .
P03.52
22p derived supernumerary marker chromosome identified
prenatally by mLPA
R. Fernandez Gonzalez1 , P. Blanco Soto1 , J. Ubeda Arades1 , B. Ferreiro1 , A.
Gonzalez de la Vega2 , M. Sanchez Hombre2 , A. Gomez Pastor3 , M. A. Orera1,2 ;
1 2 3 Hospital Gregorio Maranon, Madrid, Spain, Circagen, Madrid, Spain, Complejo
Hospitalario Universitario de Albacete, Albacete, Spain.
Supernumerary Marker Chromosomes (SMC) have a frequency of
around 0 .05% in prenatal diagnosis .Risk for congenital anomalies
ranges from 7% for SMC derived from acrocentric chromosomes to
28% for SMC derived from autosomes .
We present a prenatally detected SMC in a 32 years old woman, with
a previous spontaneous miscarriage . The ultrasound performed at 11th gestational week detected a cystic hygroma . Chorionic villus sampling
was done at that time and FISH for 13, 18, 21, XY chromosomes (Aneuvysion,
Vysys) was normal . No foetal karyotype could be obtained,
and despite normal ultrasounds during follow up, an amniocentesis
was performed at the 16th gestational week . The karyotype of the fetus
was 47,XY+mar . The marker chromosome was centromeric, satellited
and smaller than chromosome 21 . Because of the risk of Congenital
Heart Disease, we performed a FISH with TUPLE1 probe (Catch 22)
(Vysys) that was normal . Parental karyotypes were also normal .
In order to identify the origin of the SMC, we performed Multiplex Ligation
Probe Amplification (MLPA) with Salsa P070 for Telomeres (MRC-
Holland) . The result was compatible with partial trisomy of 22p . Considering
the result of FISH and MLPA, we conclude that the breakpoint
lied between the end of IL17R and the beginning of HIRA, carrying
some or all the genes associated with Cat Eye Syndrome . The parents
decided to terminate the gestation .
This case illustrates the difficulties in prenatal genetic counselling
when a SMC is involved and the need to use all technologies available
to better predict the outcomes of these pregnancies .
P03.53
Cell selection in culture of amniotic fluid cells: report of three
cases with low level of true mosaicism
N. Clusellas 1 , C. Morales 1 , I. Mademont 2 , R. Queralt 1 , A. Soler 1 , C. Badenas 1 ,
A. Sánchez 1 ;
1 Hospital Clínic de Barcelona, Barcelona, Spain, 2 CIBERER, Barcelona, Spain.
Mosaic aneuploidy is is found in 0,3% of all amniocentesis . When detected
in prenatal diagnosis is a troublesome problem on interpretation
and genetic counselling . We present three cases with low level of
mosaicism:
1 .- Twenty cells anlysed from two independent vessels showed
47,XXX, which is a good prognostic result . But a culture performed
in another laboratory showed a 45,X/47,XXX with a 75% of monosomic
cells . We revised our slides and found 7/40 of 45,X cells, which
changed the genetic counselling .
2.- The first culture showed 1/50 of 47,XY,+21 cells, the second and
the third showed 5/50 and 2/25 trisomic cells, respectively . This was
an ambiguous result, with 6% of +21 cells . We performed QF-PCR
analysis for common aneuploidies with a portion of fresh amniotic fluid
and the result was compatible with trisomy 21 .
3: The cell culture in this case was slow and poor, in order to ensure
the diagnosis, we decided to perform QF-PCR . The result was a normal
male . Finally, we could obtain some metafases for cytogenetic
analysis and the result was 37 cells with a 45,X karyotype . The gestation
was interrupted because of ultrasound findings. We repeated cytogenetics,
FISH and QF-PCR studies and the result was 45,X/46,XY .
Conclusions: There is a selection in the culture that may disturb the
real percentage of abnormal cells . Then, the percentage of abnormal
cells does not reflect the state of the fetus. The use of complementary
techniques such as QF-PCR and FISH may help to the final result.
P03.54
Identification of feto-maternal differentially methylated
regions on chromosome 18 and chromosome 21. towards the
development of fetal epigenetic markers for trisomies 18 and 21
E. Papageorgiou 1,2 , H. Fiegler 2 , N. P. Carter 2 , P. C. Patsalis 1 ;
1 The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus, 2 The Wellcome
Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United
Kingdom.
The discovery of free fetal DNA in maternal plasma has raised the
possibility for future development of noninvasive prenatal diagnosis
(NIPD) . We have been using Methylated DNA Immunoprecipitation
(MeDIP) assay and DNA oligonucleotide array technology to screen
for differentially methylated sequences between maternal blood and
placental (fetal) DNA that could be exploited for NIPD . The oligo arrays
used are specific for chromosome 18 and chromosome 21.The probes
are 50-60bp long with a median spacing of 170bp for chromosome18
and 70bp for chromosome21 .
The results have shown regions of several consecutive oligo array
probes to be differentially methylated between whole blood and placenta
. A detailed analysis has revealed 42 regions on chromosome18
and 34 regions on chromosome21 that can potentially be used as fetal
DNA epigenetic markers . The methylation status of the SERBINB5
promoter region and two additional regions on chromosome18 as well
as ten regions on chromosome21 have been confirmed by real time
quantitative PCR in six whole blood and six placental DNA samples .
Furthermore a high degree of variability has been observed between
first and third trimester placentas whereas a lower degree of variability
has been detected between individual samples of the same gestational
age . Additionally, to imitate the low percentage of fetal DNA observed
in the plasma of pregnant women, we have prepared mixtures
of 5% fetal DNA with maternal DNA and tested the enrichment of fetal
DNA by using MeDIP . Preliminary results have shown enrichment of
fetal specific methylated DNA sequences absent in whole blood DNA
samples .
P03.55
Partial trisomy 3q and monosomy 15q due to paternal
t(3;15)(q26.33;q26.1); paternal detection by m-cGH and FisH,
prenatal USG, postnatal and autopsy findings
K. Piotrowski 1 , M. Constantinou 2 , Z. Celewicz 3 , J. Patalan 4 , P. Waloszczyk 5 , S.
Zajączek 1 , B. Kałużewski 2 ;
1 Cytogenetics Unit, Szczecin, Poland, 2 Department of Medical Genetics, Medi-