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

Prenatal diagnosis
showed trisomy 21. All the results were confirmed by conventional cytogenetic.
No aneuploidies for chromosomes 13, 18, 21, X and Y were
missed by MLPA. The efficiency of the method was not influenced by
the gestational age.
MLPA is valid, simple, sensitive and cheap method of prenatal diagnosis
of common aneuploidies within 24 hours, until the complete analysis
of conventional karyotype.
P0458. A monosomy 8 cell line detected by FISH in a fetus with
multiple abnormalities and mosaic trisomy 8 in chorionic villi
D. Turchetti 1 , E. Pompilii 1 , E. Magrini 2 , A. Pession 2 , M. C. Pittalis 3 , D. Santini 4 ,
P. Bonasoni 4 , M. Segata 3 , G. Pilu 3 , G. Romeo 1 , M. Seri 1 ;
1 Unit of Medical Genetics, University of Bologna-Policlinico S.Orsola-Malpighi,
Bologna, Italy, 2 Department of Oncology-Section of Pathology, University of
Bologna-Ospedale Bellaria, Bologna, Italy, 3 Department of Obstetrics and
Gynecology, University of Bologna-Policlinico S.Orsola-Malpighi, Bologna,
Italy, 4 Unit of Pathology, University of Bologna-Policlinico S.Orsola-Malpighi,
Bologna, Italy.
Trisomy 8 mosaicism is associated with a variable phenotype, with
neurodevelopmental delay, dysmorphic facial features, skeletal, renal
and cardiovascular abnormalities being common features. Nevertheless,
individuals with minor abnormalities and normal intelligence have
been described.
Increased nuchal translucency thickness was detected at 13 weeks
in the male fetus of a 27-year-old woman. At CVS, direct preparation
showed a normal karyotype (46,XY), while in cultured cells mosaic trisomy
8 (47,XY,+8[11]/46,XY[53]) was found. At 15 +6 weeks, ultrasound
scan revealed bilateral cleft lip and palate with flat face and absence
of the nose; no other abnormalities were evidenced. Pregnancy was
terminated at 16 +6 weeks.
Mosaic trisomy 8 was confirmed by standard karyotyping in placenta
(47,XY,+8[13]/46,XY[87]) and amnion (47,XY,+8[16]/46,XY[68]), but
not in umbilical cord (46,XY[100]). Pathological examination of the fetus
confirmed cleft lip and palate with maxillary hypoplasia and showed
gastrointestinal abnormalities, dysplastic kidneys and focal portal fibrosis
in the liver. To confirm the presence of the trisomic cell line in
fetal tissues, FISH was performed in liver and kidney samples using
a chromosome-8 specific probe. In liver, two fluorescent signals were
detected in 64% of nuclei, three signals in 13%, one signal in 23%; in
kidney, 57% of nuclei showed two signals, 43% one signal. In control
tissues, the presence of one signal was found in 3-10% of nuclei, suggesting
that our finding reflected true mosaic monosomy 8.
In the case here described, multiple fetal anomalies were associated
with a complex chromosomal mosaicism (trisomy/monosomy 8) with
only trisomy 8 mosaicism detected at CVS.
P0459. Maternal MTHFR genotype, blood homocysteine levels
and incidence of NTDs and Down syndrome
S. Andonova 1,2 , V. Dimitrova 3 , R. Vazharova 4 , R. Tincheva 5 , A. Tzoncheva 6 , I.
Kremensky 1,2 ;
1 Molecular Medicine Center, Sofia Medical University, Sofia, Bulgaria, 2 National
Genetics Laboratory, University Hospital of Obstetrics and Gynecology, Sofia,
Bulgaria, 3 High-Risk Pregnancy Department, University Hospital of Obstetrics
and Gynecology, Sofia, Bulgaria, 4 Department of Medical Genetics, Sofia
Medical University, Sofia, Bulgaria, 5 Section of Clinical Genetics, Department
of Pediatrics, Sofia Medical University, Sofia, Bulgaria, 6 Department of Clinical
Laboratory and Clinical Immunology, Sofia Medical University, Sofia, Bulgaria.
Neural Tube defects (NTDs) and Down syndrome (DS) are common
reason for malformations among newborns. Despite of the high frequencies,
the etiology is still unclear. Some anomalies in the homocysteine
metabolism and elevated blood homocysteine levels in mothers
in combination with folate deficiency could be associated with NTD
and DS appearance. The C677T and A1298C polymorphisms in Methylenetetrahydrofolate
reductase (MTHFR) gene were described as an
NTD risk factor and could cause elevated homocysteine concentrations.
The frequency of C677T and A1298C substitutions were detected
after restriction of the PCR products with HinfI and MboII, respectively.
We have investigated 54 DNA samples from NTD mothers, 35 -
from DS mothers and 60 - from control mothers. Plasma homocysteine
levels were measured by chemiluminescence immuno assay. There
was no statistical difference in allele frequencies according C677T.
The observed frequencies of 1298C allele were 37.0%, 26.9% and
28.8% respectively. The frequency of AC genotype was statistically dif-
ferent between DS mothers and control and NTD mothers. Statistical
differences were registered also between DS mothers and NTD mothers,
regarding AA genotype. The measured mean total homocysteine
level in the group of 49 pregnant control females was 3,14 umol/L, in
comparison with the group of 11 women with NTD pregnancy (mean
10,2 umol/L). The data presented in this study failed to support the
relation between MTHFR 677C>T and 1298A>C polymorphisms and
risk of having a child with NTD and DS. Raised plasma homocysteine
levels could be explained by folic acid deficiency, mutations in MTHFR
genes or both.
P0460. Identification and characterization of DNA methylation
sensitive markers for non invasive diagnosis of X linked
aneuploidies.
F. Della Ragione, L. Speranza, M. D’Esposito;
Institute of Genetics and Biophysics, Naples, Italy.
We are interested to the development of NIPD methodogies of X
linked aneuploidies based on DNA methylation. We identified three
new markers by “in silico” strategies. Among these markers, two are
genes escaping X inactivation, and predicted to be expressed only in
placenta; a third gene is X inactivated and expressed only in placenta
as well. We next analyzed their expression by RT-PCR in placenta and
blood: on this basis, the first, escaping gene was eliminated, given its
expression in both tissues. The remaining analysis has been focused
on the remaining two genes.
We confirmed their inactivation status, by somatic hybrids analysis:
as predicted one gene is escaping, the other is subject to X inactivation.
By bisulfite analsysis we demonstrated that the escaping gene is
differentially methylated between blood and placenta, whereas the X
inactivated gene, being not regulated by differential DNA methylation
has been rejected.
Additional efforts will be necessary to complete the characterization of
the marker we identified, and we will continue the search for additional
markers. We plan to develop an MSP assay to check the differential
methylation of these regulatory regions in blood and in placenta: later
on, a Real Time PCR assay to determine the number of X and Y chromosomes
of a certain sample. Our final goal is to adopt this strategy on
plasma of donors for non invasive diagnosis of X linked aneuploidies.
This research is sponsored by CEE: SAFE: Special Non-Invasive Advances
in Foetal and Neonatal Evaluation Network, contract LSHB-
CT-2004-503243.
P0461. Foetal sex assessment in maternal plasma in the first
trimester of gestation: large scale validation of the technique for
clinical purposes.
A. Bustamante-Aragones 1 , M. Rodriguez de Alba 1 , M. J. Trujillo-Tiebas 1 , J.
Plaza 2 , R. Cardero-Merlo 1 , F. Infantes 1 , C. Gonzalez-Gonzalez 1 , M. L. Perez 2 ,
C. Ayuso 1 , C. Ramos 1 ;
1 Department of Genetics. Fundacion Jimenez Diaz-Capio-CIBER-ER(ISCIII),
Madrid, Spain, 2 Department of Obstetrics & Gynaecology. Fundacion Jimenez
Diaz-Capio, Madrid, Spain.
The knowledge of the foetal gender is an indispensable data for those
couples at risk of an X-linked disorder. The possibility to determine the
foetal sex from a maternal plasma sample collected in the early first trimester
of gestation would avoid invasive prenatal procedures in some
cases. For this reason, we have analyzed a large number of maternal
plasma samples collected in the first trimester of gestation in order to
know the earliest accurate gestational age to perform the study. The
next step will be the application of this technique for clinical purposes.
Up to date, 185 voluntary pregnant women from the 5 th to 12 th week of
gestation have participated in this study, having collected a total of 278
samples. Three replicas of each sample were analyzed by RealTime-
PCR and the foetal gender assessment was established based on the
presence/absence of the SRY gene. Three ways to validate results are
being used: 1) By the analysis of a second blood sample within the first
trimester. 2) By comparing with the prenatal tests results (CVS, amniocentesis
or 20 th week ecography). 3)By comparing both a second
sample + prenatal tests.
At the present moment, results from 138 out of 185 pregnant women
have been confirmed obtaining a 100% of accuracy and specificity in
samples collected from the 6 th to the 12 th week of gestation. Although
the study has not finished yet, these results indicate the possible immediate
application of the technique for clinical diagnosis in our hospital.
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