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

Concurrent Sessions
port the findings of this screen, including SNPs that are significantly
associated with increased risk of bowel cancer. These SNPs are now
being tested in a phase 2 study involving 3,000 unselected cases and
3,000 controls. Further collaborative studies involving large sample
sets will be needed to confirm that SNPs from our phase 1 and 2 data
are truly associated with disease.
C53. Hereditary Diffuse Gastric Cancer (HDGC) patients and
CDH1 mutations: a systematic review of the literature
C. Oliveira 1 , R. Karam 1 , C. Graziadio 2 , H. Pinheiro 1 , S. Sousa 1 , R. Seruca 1,3 ;
1 Institute of Molecular Pathology and Immunology of the University of Porto,
Porto, Portugal, 2 Department of Genetics, Fundação Faculdade Federal de
Ciências Médicas de Porto Alegre (FFFCMPA), Porto Alegre, Brasil, Porto
Alegre, Brazil, 3 Medical Faculty of the University of Porto, Porto, Portugal,
Porto, Portugal.
Worldwide, gastric cancer is the second cause of cancer-related
death. Despite an overall trend for decrease, incidence of gastric
cancer in young patients and cases with familial clustering remains
stable. Despite being an uncommon disease, HDGC is a major health
problem and extremely difficult to address in clinical and therapeutic
grounds, due to its severity and unavailability of early diagnosis. A
single gene was identified with a causative role in HDGC, E-cadherin.
We performed a systematic review of CDH1 mutation carriers clinical
presentation and its association with frequency, localization and
type of CDH1 germline mutations. We collected information on 99
CDH1 mutation carriers, described to date in the literature. The age
of onset of CDH1 mutation carriers varied between 16 and 73 years
old (mean=41.8±14.4), and 75% of cases were diagnosed before
50. Male:female ratio was near 1.0. Although CDH1 mutations were
distributed along all gene sequence, exons 2,3,7 and 11 were preferentially
affected. Most mutation carriers (87.9%-87/99) harboured
CDH1 truncating mutations. The type of mutations (truncating and missense)
was significantly different between families with complete and
incomplete criteria for HDGC, previously established by the IGCLC
(p=0.0001). Carriers from families with complete criteria harboured
preferentially truncating mutations (92%), while carriers from families
with incomplete criteria harboured truncating and missense mutations
in similar frequencies (50%). Moreover, truncating mutations carriers
were significantly younger (40.5±14.3) than missense mutation carriers
(50.7±12.4) (p=0.02). This systematic analysis is of crucial importance
to help genetic counselling and to direct molecular analysis in
suspected HDGC mutation carriers.
C54. The identification of (ETV6/)RUNX1-regulated genes in
lymphopoiesis using histone deacetylase inhibitors in ETV6/
RUNX1-positive lymphoid leukaemic cells
J. Starkova 1 , J. Madzo 1 , G. Cario 2 , T. Kalina 1 , A. Ford 3 , M. Zaliova 1 , O. Hrusak 1 ,
J. Trka 1 ;
1 CLIP – Childhood Leukaemia Investigation Prague, Department of Paediatric
Haematology and Oncology, Charles University, 2nd Medical School, Prague,
Czech Republic, 2 Department of Paediatrics, University Hospital Schleswig-Holstein,
Kiel, Germany, 3 Leukaemia Research Fund Centre, Institute of Cancer
Research, Chester Beatty Laboratories, London, United Kingdom.
Chimeric transcription factor ETV6/RUNX1 (TEL/AML1) is believed to
cause pathological block in lymphoid cells development via interaction
with corepressor complex and histone deacetylase. We wanted
to demonstrate regulatory effect of ETV6/RUNX1 and its reversibility
by histone deacetylase inhibitors (HDACi) and to identify potential
ETV6/RUNX1-regulated genes. We used luciferase assay to demonstrate
the interaction of ETV6/RUNX1protein, ETV6/RUNX1-regulated
gene and HDACi. In order to identify ETV6/RUNX1-regulated genes
we employed expression profiling and HDACi in the lymphoid cells.
Next using the flow cytometry and qRT-PCR we measured changes in
gene and proteins expression after HDACi treatment. Luciferase assay
showed repression of granzyme B expression by ETV6/RUNX1
protein and reversibility of this effect by HDACi. Proving this regulatory
role of ETV6/RUNX1, we used complex statistical analysis to
identify 25 genes that are potentially regulated by ETV6/RUNX1 protein.
In 4 selected genes with known role in the cell cycle regulation
(JunD, ACK1, PDGFRB and TCF4) we confirmed expression changes
after HDACi by quantitative analysis. After HDACi treatment, ETV6/
RUNX1-positive cells showed immunophenotype changes resembling
differentiation process compared to other leukaemic cells (BCR/ABL,
ETV6/PDGFRB-positive). Moreover, ETV6/RUNX1-positive leukaemic
cells accumulated in G1/G0 phase after HDACi while other B-lineage
leukaemic cell lines showed rather unspecific changes including induction
of apoptosis and decreased proliferation. Presented data support
the hypothesis that HDACi affect ETV6/RUNX1-positive cells via direct
interaction with ETV6/RUNX1 protein, and that treatment with HDACi
may release aberrant transcription activity caused by ETV6/RUNX1
chimeric transcription factor.
Supported by grants IGA MZ 8316 and MSM0021620813.
C55. Analysis of the genomic insertion sites of viral gene
therapy vectors using next generation sequencing technologies.
C. Bauser1 , M. Schmidt2 , C. von Kalle2 ;
1 2 GATC Biotech, Konstanz, Germany, National Center for Tumor Diseases
(NCT), Heidelberg, Germany.
Viral vectors commonly used for somatic gene therapy can cause insertional
mutagenesis, activating oncogenes in the patient, potentially
leading to oncogenic transformation. After transduction, the viruses integrate
into the genome, preferentially upstream of actively expressed
genes. The potential therefore exists to alter the expression of oncogenic
or tumor suppressor genes which occasionally leads to changes
in stem cell self-renewal and oncogenic transformation. Clonal expansion
of a cell with an integration event that promotes cell division, and
possibly oncogenic transformation, leads to the dominance of a few (or
single) integration events in hematopoietic progenitor cells.
By using LAM-PCR, DNA-sequencing and bioinformatic analysis, we
can identify the viral integration sites in the genome. Because the integration
site can differ in each cell, it can be difficult to identify all genes
potentially influenced by the integration events. Whereas cloning and
Sanger sequencing only allow identification of several hundred integration
events, next generation sequencing methods allow the identification
of ten thousands of individual integration events in parallel.
The ability to identify more integration events early in the gene therapy
treatment procedure, and follow changes in the distribution of cells
having different integration sites, will allow to better assess the safety
of somatic gene therapy, and develop new and safer viral vectors and
transduction methods.
We present the results of a systematic insertion site analysis for a
retroviral vector and show that the broad range of insertion sites found
using next generation sequencing technology is a vast improvement
over the traditional Sanger method.
C56. Correction of VLCAD deficiency and prediction of mutation
severity with bezafibrate: how to kill two birds with one stone.
S. Gobin-Limballe 1 , F. Djouadi 1 , F. Aubey 1 , S. Olpin 2 , S. Yamaguchi 3 , R. Wanders
4 , T. Fukao 5 , J. Kim 6 , J. Bastin 1 ;
1 CNRS UPR 9078, Paris, France, 2 Sheffield Children’s Hospital, Sheffield,
United Kingdom, 3 Shimane School of medicine, Shimane, Japan, 4 Academic
Medical Center, Amsterdam, The Netherlands, 5 Gifu University, Gifu, Japan,
6 Medical College of Wisconsin, Milwaukee, WI, United States.
We recently showed that fibrates could restore FAO in patient cells harboring
inborn defects in Very-Long-Chain-AcylCoA-Dehydrogenase (VL-
CAD; mitochondrial β-oxidation), by stimulating residual enzyme activity.
Given the variety of reported VLCAD gene point mutations, we investigated
the response to drug as a function of genotype. 34 VLCAD-deficient fibroblast
with distinct genotypes representing 50 different mutations were
treated with 400µM bezafibrate for 72h and FAO was measured using
tritiated palmitate. Untreated cells exhibited FAO rates much lower (-30
to -90%) than control. Bezafibrate induced a marked increase in FAO in
60% of the genotypes tested, and a complete correction in 15 cell lines.
These data allowed to identify three groups: - severely deficient cells
with nonsense mutations, or missense mutations affecting residues essential
for catalysis (G222, G441, R469), that were drug-resistant -a 2nd
group with missense mutations compatible with a moderate response to
bezafibrate - a 3rd group which harbored genotypes compatible with a
full restoration of FAO by bezafibrate, pointing to mild mutations (V283A,
G441D, R615Q). We also characterized changes in VLCAD mRNA and
residual enzyme activity levels induced by bezafibrate, as a function of
genotype. The mutations were reported in a predictive VLCAD 3-D model
allowing to confirm the mild or severe mutations that were characterized
in the “bezafibrate test”. The response to bezafibrate can therefore
predict the severity of VLCAD point mutations that was not documented
yet and might help to identify patients for a future clinical trial.
2