2008 Barcelona - European Society of Human Genetics

Concurrent Sessions
and class 4 embryo group . For the diagnostic value, Positive- (PPV)
and Negative Predictive Value (NPV) were calculated .
In our centre 80 women underwent PGD-PCR, resulting in 793 embryo
genotypes, 241 unaffected embryos were used for ET . PGD-PCR
blastomere outcome, scored as affected or aberrant in 234/241 positive
embryos (Se; 97,1%), and scored unaffected in 181/206 negative
embryos (Sp: 87,9%) . Out of the 7 false negative embryos, 6 were
graded as class 4 . The Se in the class 4 embryo group was 90,2% and
Sp 93,2% . Exclusion of class 4 embryos resulted in Se of 99,4%, Sp of
86,4% and LR positive test of 7 .3 and LR negative test of 0 .006 . The
PPV of an abnormal PGD-PCR is 89 .1%, the NPV of a normal PGD-
PCR is 99 .3% in this group .
PGD-PCR procedure is validated as a diagnostically reliable method
for selecting unaffected embryos for ET . Accuracy of PGD-PCR analysis
improves by rejecting class 4 embryos for ET .
c09.5
Multiple displacement amplification (MDA) in preimplantation
genetic diagnosis (PGD)
M. De Rycke 1,2 , C. Spits 2 , W. Verpoest 3 , K. Sermon 2 , J. Vanderelst 3 , I. Liebaers
1,2 ;
1 Centre for Medical Genetics UZ Brussel, Brussels, Belgium, 2 Department of
embryology and genetics Vrije Universiteit Brussel, Brussels, Belgium, 3 Centre
for Reproductive Medicine, UZ Brussel, Brussels, Belgium.
Introduction
PGD is an alternative to prenatal diagnosis, performed on single blastomeres
from in vitro cultured embryos . Since many of the PGD requests
for single-gene disorders involve new developments and the workup of
single-cell multiplex-PCR is technically demanding and requires time
and manpower, we evaluated a different approach relying on singlecell
whole genome amplification (WGA) using MDA as a universal first
step, followed by regular PCRs for specific loci.
Materials and Methods
During pre-clinical workup, MDA products from single lymphoblasts
were used to assess the amplification efficiency, preferential amplification
(PA), allele drop out (ADO) and contamination rates of loci
(specific mutations and linked STR markers) involved in cystic fibrosis,
Marfan syndrome, Huntington’s disease, spinal cerebral ataxia 7 and
neurofibromatosis type 1.
Nine couples underwent 18 clinical PGD cycles, in which 100 embryos
were biopsied .
Results
The pre-clinical results showed an amplification efficiency of 96%; no
contamination was detected . This is similar to PCR-based protocols .
The PA and ADO rates varied for the different loci and the average rate
of 25% was five fold higher than with PCR. Still, the diagnostic efficiency
in the clinical cycles was 93 % . Twenty embryos were transferred
in 13 cycles, resulting in two biochemical pregnancies, one singleton
baby, one twin and one pregnancy ongoing .
Conclusions
The relatively high ADO and PA rates associated with single-cell MDA
in PGD was overcome by analysis of at least four loci . The diagnostic
efficiency is similar to PCR-based protocols. These results prove the
applicability of single-cell MDA in PGD .
c09.6
Fluorescence in-situ Hybridisation using Oligonucleotide
probes (Oligo-FisH): a new strategy for Preimplantation Genetic
screening (PGs).
L. Robertson1 , S. Doubravska1 , M. Grigorova1 , D. K. Griffin2 , A. H. Handyside1 ,
A. R. Thornhill1 ;
1 2 Bridge Genoma, London, United Kingdom, University of Kent, Canterbury,
United Kingdom.
The principle of preimplantation genetic screening (PGS) is to biopsy 1
cell from a 6-10-cell embryo and determine chromosomal copy number
within a 24 hr time frame before transferring chromosomally ‘normal’
embryos to the patient . Current PGS consists of sequential multicolour
FISH using two or more probe sets derived from BACs . Our current
clinical method consists of two rounds of FISH as follows: (1) chromosomes
13, 16, 18, 21 and 22 with a 2 .5hr hybridisation time and 2) X,
Y, 15 with a 16-18 hr hybridisation time allowing results to be reported
in 24hr facilitating Day 4 embryo transfer . Recently FISH probes using
labelled 30-mer oligonucleotides (ODNs) have been developed which
specifically hybridise to repetitive sequences on a range of different
chromosomes . Based on the manufacturer’s protocol, with a hybridisation
step of 5 minutes, PGS for 8 chromosomes (2 x 4 chromosome
probe sets) was performed in 1 hour . Four different ODNs probe sets
for chromosomes (1) X, Y, 15,17 (2) X, Y, 16 (3) X, Y, 16q and (4) X, Y,
18 were validated using known normal donor lymphocytes . Validation
of ODNs probe sets for chromosomes X, Y, 15,17; X, Y, 16; X, Y, 16q
and X, Y, 18 scored >80% for probe efficiency using a short hybridisation
time of 5 minutes. With modifications, we expect to reach > 95%
efficiency for each probe set. The short length of ODN probes permits
rapid hybridisation, a significant advantage for time critical procedures
such as PGS .
c10.1
BRcA1 mutations and prostate cancer in Poland
J. Lubinski1 , C. Cybulski1 , B. Gorski1 , J. Gronwald1 , T. Huzarski1 , T. Byrski1 ,
T. Debniak1 , A. Jakubowska1 , D. Wokolorczyk1 , B. Gliniewicz1 , A. Sikorski1 , M.
Stawicka2 , D. Godlewski2 , Z. Kwias3 , A. Antczak4 , K. Krajka5 , W. Lauer6 , M. Sosnowski7
, P. Sikorska-Radek8 , K. Bar9 , R. Klijer10 , Z. Romuald1 , B. Malkiewicz11 ,
A. Borkowski7 , T. Borkowski9 ;
1 2 Pomeranian Medical University, Szczecin, Poland, Prophylactic and Epidemiology
Center, Poznan, Poland, 3Medical University in Poznan, Poznan, Poland,
4 5 Medical University of Lodz, Lodz, Poland, Medical University of Gdansk,
Gdansk, Poland, 6Institut für Biomedizinische Technologien, Aachen, Germany,
7 8 Medical University, Warsaw, Poland, Medical University, Poznan, Poland,
9 10 Medical University, Lublin, Poland, University Hospital of Lublin, Lublin, Poland,
11Medical University, Wroclaw, Poland.
Evidence to date that BRCA1 mutation carriers are at an increased risk
of prostate cancer is mixed - both positive and negative studies have
been published . To establish whether inherited variation in BRCA1 influences
prostate cancer risk we genotyped 1793 men with prostate
cancer in Poland and 4570 controls for three founder mutations (C61G,
4153delA, 5382insC) . BRCA1 mutation was present in 0 .45% of cases
and 0 .48% of controls (OR=0 .9; P=1 .0) . The odds ratios varied substantially
by mutation . The 5382insC mutation is the most common of
the three founder mutations . It was detected only in one case (0 .06%),
whereas it was seen in 0 .37% of controls (P=0 .06) . In contrast, the
4153delA was more common in prostate cancer cases (0 .22%) than in
controls (0.04%) (OR=5.1; 95% confidence interval: 0.9-27.9; P=0.1).
The C61G mutation was also found in excess in cases (0 .17%) compared
with controls (0.07%) (odds ratio=2.6; 95% confidence interval:
0 .5-12 .7; P=0 .5) . Eight men with prostate cancer carried a mutation .
Only one of these carried the 5382insC mutation, compared with 17
of 22 individuals with mutations in the control population (P=0 .003) .
These data suggest that 5382insC mutation is unlikely to be pathogenic
for prostate cancer in Polish population . The presence of one of the
other alleles was associated with an increased risk for prostate cancer
(OR=3.6; 95% confidence interval: 1.1-11.3; P=0.045); in particular
for familial prostate cancer (OR=12; 95% confidence interval: 2.9-51;
P=0 .0004) . We consider that the risk of prostate cancer in BRCA1 carriers
varies with the position of the mutation .
c10.2
Genomic differences between retinoma and retinoblastoma
M. Amenduni 1 , K. Sampieri 1 , F. Ariani 1 , M. Bruttini 1 , M. A. Mencarelli 1 , M. C.
Epistolato 2 , P. Toti 2 , S. Lazzi 2 , A. Marozza 1 , F. Mari 1 , T. Hadjistilianou 3 , S. De
Francesco 3 , A. Acquaviva 4 , A. Renieri 1 ;
1 Medical Genetics, Siena, Italy, 2 Department of human pathology and oncology,
Siena, Italy, 3 Retinoblastoma referral center,Department of ophtalmology, Siena,
Italy, 4 Department of pediatrics,obstetrics and reproductive medicine,italian
retinoblastoma registry, Siena, Italy.
Genomic copy number changes are involved in the multi-step process
transforming normal retina in retinoblastoma (RB) after RB1 mutational
events . Previous studies on tumor samples led to a multi-step model
in which after two successive RB1 mutations, the following genomic
changes accompany malignancy: 1q32 .1, 6p22 and 2p24 .1 gains and
16q22 losses . Recent data have demonstrated that retinoma, a rare
benign retinal lesion, represents an early stage in the pathway to RB .
In order to catch somatic events that determine retinoma-RB transition,
we investigated genomic copy number changes in DNA isolated by laser
capture from retinoma and retinoblastoma tissues of two different
patients . We employed both genome wide array-CGH technique and
Real-Time qPCR at four genes involved in RB pathogenesis (MDM4,