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

Prenatal diagnosis
Po03. Prenatal diagnosis
P0429. Reporting Three Cases Of PND For Alpha thalassemia In
Iran.
V. Lotfi 1,2 , M. Sajedifar 1,2 , P. Fouladi 1,2 , A. Abdolhosseini 1 , F. Shirazi 1 , Z. Shahab
1 , S. Zeinali 1,3 ;
1 Medical Genetics Lab of Dr Zeinali, Tehran, Islamic Republic of Iran, 2 Young
Researchers Club of research and science branch of Islamic Azad University,
Tehran, Islamic Republic of Iran, 3 Human Genetics Unit, Dept of Biotech ,Pasteur
Institute, Tehran, Islamic Republic of Iran.
Each normal person has 4α genes which are shown as αα /αα. Usually
α 2 is more expressed than α 1 therefore deletion of α 2 or mutation on α 2
has more severe effect.
Being carrier alpha thalasemias are usually caused by deletion of one
or two α-globins genes.
Alpha thal genotyped are usally shown like -α/αα (αthal 2); -α /-α or
--/αα (α thal 1 or classical type). --/αα type is seen most in South East
Asia. H-disease is caused by
--/-α form either by deletions or point mutations (e.g. --/α T α ).
Complete absence of 4α genes causes hydrops fetal is which requiring
prenatal diagnose. We have performed three cases of PND for α-thal
so far. In these cases the fetuses were diagnosed carriers (--/αα). In
one case both parents were carrier of Med deletion (-- med /αα). The
Hematological data of parents were:
Father: MCV=62/8 MCH=20/2 Hb=14/1 A 2 = Normal
Mother:MCV=67/9 MCH=20/6 Hb=10/3 A 2 =Normal
At the present time we think only need to perform PND for hydrops
fealties.
Yet we need more strong evidence to do PND since most cases of
Hdisease seen in adults by us were mild enough to exclude PND for
similar cases.
Key words: Alpha thalassemia, Prenatal Diagnosis, H Disease
P0430. Whole-genome expression analysis of amniocytes
G. R. Nagy 1 , B. Győrffy 2 , O. Galamb 3 , B. Nagy 1 , L. Lázár 1 , Z. Bán 1 , B. Molnár 3 ,
Z. Papp 1 ;
1 Semmelweis University, I. Department of Obstetrics and Gynecology, Budapest,
Hungary, 2 Semmelweis University, Szentágothai János Knowledge
Center, Budapest, Hungary, 3 Semmelweis University, II. Department of Internal
Medicine, Cell Analysis Laboratory, Clinical Research Unit of the Hungarian
Academy of Sciences, Budapest, Hungary.
Aims: The objective of our study was to investigate the ability of global
gene expression array analysis from routinely collected amount of amniotic
fluid. We also wanted to search for the differential gene expression
profile of a polygenic disorder, the neural tube defect, which is the
second most common birth defect in the world.
Material and methodology: We analyzed 6-17 mL of amniotic fluid.
Samples were taken from seven pregnant women carrying fetuses with
neural tube defect, diagnosed during ultrasound examination. Control
samples were obtained from pregnant women who underwent routine
genetic amniocentesis because of advanced maternal age (>35
years). Fetal mRNA from amniocytes was successfully isolated, amplified,
labeled, and hybridized to whole-genome transcript arrays. Since
the most significant epidemiological finding with respect to neural tube
defects is the protective effect of maternal periconceptional folic acid
supplementation, we also investigated specific folate-related genes.
Results: The detected differential gene expression profiles between
cases and controls highlighted genes, like SLA, LST1 and BENE
genes, these might be important in the development of neural tube
defects. None of the specific folate-related genes were in the top 100
associated transcripts.
Conclusions: This study demonstrates the ability of global gene expression
array analysis from as small as 6 ml amniotic fluid, allowing
the examination from routinely collected amniotic fluid samples so individual
fetuses can be studied. The analysis of fetal gene expression
differences might get us closer to decipher the complex genetic background
of polygenic disorders.
P0431. Preimplantation genetic diagnosis for autosomal
recessive polycystic kidney disease
N. Gigarel 1 , N. Frydman 2 , P. Burlet 1 , E. Feyereisen 2 , V. Kerbrat 2 , R. Frydman 2 ,
J. Bonnefont 1 , A. Munnich 1 , J. Steffann 1 ;
1 Département de génétique, Necker, Paris, France, 2 Biologie de la reproduc-
1 0
tion, Béclère, Clamart, France.
Autosomal Recessive Polycystic Kidney disease (ARPKD) is one
of the most common hereditary renal cystic disease, and is caused
by mutations in the PKHD1 gene. The diagnosis is often made late
in pregnancy and sometimes at birth. Owing to the poor prognosis,
there is a strong demand for prenatal diagnosis. Preimplantation genetic
diagnosis (PGD) represents an alternative because it is done on
cells taken from in vitro fertilized embryos at the third-day stage. Only
healthy embryos are transferred, avoiding the physical and psychic
traumatism of the termination of pregnancy in the case of an affected
fetus detected later by prenatal diagnosis. We developed a single-cell
diagnostic approach based on haplotype analysis in order to propose
PGD to most couples, whatever the mutations may be.
Six linked markers within (D6S1714 and D6S243), or in close proximity
to (D6S272,D6S436, KIAA0057, D6S1662), the PKHD1 gene are
tested in multiplex nested-PCR, using QIAGEN multiplex PCR kit, allowing
identification of embryos carrying the high-risk haplotypes.
PCR analysis was first carried out on 50 single-lymphocytes. Amplification
rate was excellent (100%), with an allele drop-out (ADO) rate
ranging from 0 to 8%. Using this test, 4 PGD cycles were performed
resulting in 18 biopsied embryos. Transferable embryos were obtained
for three cycles, resulting in a pregnancy, and the birth of a healthy boy.
The co-amplification of several loci increases the assay accuracy by
allowing the detection of ADO, recombination and contamination. This
simple diagnostic procedure can be applied to most patients at risk to
transmit ARPKD.
P0432. Non Invasive Prenatal Diagnosis (NIPD): the suitability
of locked nucleic acid (LNA)-modified allele-specific primers for
detection of paternally inherited beta globin gene alleles
G. Dimisianos, J. Traeger-Synodinos, C. Vrettou, E. Kanavakis, A. Mavrou;
University of Athens, Athens, Greece.
Prenatal diagnosis (PND) with CVS sampling and amniocentesis involves
a small risk of pregnancy complications. This may be avoided by
NIPD, which for monogenic diseases is potentially based on detection
of distinct paternally-inherited alleles of cell-free fetal DNA in the maternal
circulation. However, methods for NIPD must address the problem
that fetal alleles exist within an excess of maternal alleles (~5%
versus ~95%) with low total-DNA concentration (~10-100pg/μl). This
study investigates specificity and sensitivity of allele-specific PCR using
LNA-modifed oligonucleotides for the most common Mediterranean
beta-thalassaemia mutation (IVS-I-110G->A, HBB c.93+21G>A) when
present in minority quantities relative to wild-type alleles (20%:80%).
Design of PCR primers included an allele-specific primer (IVS1-110
G>A), LNA-modified at the 3’ nucleotide (mutant base). Inclusion of a
pBR322 RsaI-fragment and pBR322-specific primers monitored PCR
conditions whilst overcoming the problem of co-amplifying genomic regions
present in excess from the maternal genome. Reverse primers
were labelled (Cy5.5), facilitating analysis on an automatic sequencer.
PCR reactions analysed 200 diluted genomic-DNA mixtures with 80%
normal and 20% mutant alleles (M/N), and 100 mutation-free (N/N)
DNA samples (8-80pg DNA /reactions). 32 M/N and 32 N/N DNA samples
were also analysed with non-LNA allele-specific primers under
identical conditions. LNA-modified primers gave 2% false positives
and no false negatives; non-modified primers gave false positive and
negative rates >40%. The pBR322-specific band confirmed correct
PCR conditions in all cases. These results indicate that LNA-modified
allele-specific primers increase specificity and sensitivity.
P0433. Prenatal diagnosis of Carpenter Syndrome
D. F. Albu, A. C. Crenguta, E. Severin;
“Carol Davila” Univ Med Pharm, Bucharest, Romania.
Background: Carpenter syndrome (CS) is a rare genetic disorder also
called acrocephalopolysyndactily. The main features of Carpenter syndrome
are craniosynostosis, congenital heart disease, obesity, extra
fingers and/or toes (polydactyly), genital abnormalities, and short stature.
Carpenter syndrome is an autosomal recessive disorder, for which
the gene has not yet been identified. Objectives: To identify and characterize
both craniofacial and limbs malformations associated with CS.
Patients and Methods: A 28-year-old pregnant Caucasian female was
referred at 21 weeks’ gestation for a routine prenatal ultrasound. Fetal
monitoring was made by ultrasound scans for fetal growth, congenital
malformations, and amniotic fluid volume. We also collected informa-