2008 Barcelona - European Society of Human Genetics

Cancer genetics
and/or prognostic markers, human cell lines and tissues from mice
treated with DEHP and BF .
β-catenin and APC gene mutational analysis, modulation of gene
expression analysis, genomic post-trascriptional regulation analysis
through microRNA and protein expression patterns are in progress .
We assessed i) the in vitro cytotoxicity of DEHP and BF in the HB cell
line HuH6, ii) the in vitro differential expression of two microRNAs, iii)
the differential protein expression in some gene products of the Wnt/βcatenin
signalling pathway .
Project funded in the frame of ISS-NIH collaboration “Tackling rare diseases
yet lacking diagnosis and/or prognosis: a pilot integrating data
collection and experimental studies” (2006)
P04.154
Influence of the focal adhesion protein leupaxin on invasion and
metastasis in tRAmP prostate carcinomas
S. Beckemeyer1 , S. Schweyer2 , P. Burfeind1 , S. Kaulfuß1 ;
1 2 Institute of Human Genetics, Göttingen, Germany, Department of Pathology,
Göttingen, Germany.
In a recent study we showed that leupaxin expression could be detected
in 21% of human prostate carcinomas (PCa) . Down-regulation
of leupaxin expression using RNAi resulted in apoptosis of more than
50% of androgen-dependent LNCaP PCa cells and in a significant reduction
of invasiveness and migratory ability of androgen-independent
PC-3 and DU145 PCa cells .
To analyse the role of leuapxin in progression and invasion of PCa
in vivo we bred transgenic mice expressing prostate-specific leupaxin
with TRAMP mice to generate double transgenic heterozygous mice .
The transgenic adenocarcinoma of the mouse prostate (TRAMP) model
develops, as a consequence of transgene SV40 T/t antigen expression,
progressive prostate cancer that is invasive and capable of metastatic
spread . Histopathological analysis showed that at the age of 22
weeks 91% of double heterozygous mice display a poorly differentiated
tumor . In addition, 65% of these mice developed metastasis to the
lymph nodes . In single heterozygous TRAMP mice only 50% showed
a well differentiated tumor while none of them developed metastasis .
Furthermore, our recent studies could demonstrate that the expression
of the cell-cell-adhesion molecule catenin delta 1 (p120CTN)
negatively correlated with the expression of leupaxin . No expression
of p120CTN could be detected in progressive and invasive prostate
tumors of double transgenic TRAMP/leupaxin mice . In conclusion, the
results of our studies indicate that leupaxin expression enhances the
progression of PCa towards a more aggressive and metastatic tumor
and that the loss of p120ctn expression due to leupaxin overexpression
represents one possible cause of the invasive behavior .
P04.155
Does nonsense-mediated RNA decay support tP53
haploinsufficiency as the cause of tumour development in Li-
Fraumeni syndrome?
K. Prochazkova 1 , M. Mrhalova 2 , A. Augustinakova 2 , D. Sumerauer 3 , A. Puchmajerova
1 , R. Kodet 2 , Z. Sedlacek 1 ;
1 Department of Biology and Medical Genetics, Charles University - Second
Medical School and University Hospital Motol, Prague, Czech Republic, 2 Department
of Pathology and Molecular Medicine, Charles University - Second
Medical School and University Hospital Motol, Prague, Czech Republic, 3 Department
of Paediatric Haematology and Oncology, Charles University - Second
Medical School and University Hospital Motol, Prague, Czech Republic.
Li-Fraumeni syndrome (LFS) is characterised by predisposition to a
broad spectrum of cancers . The syndrome is associated with germline
TP53 mutations, usually missense . The aberrant protein produced from
the mutated TP53 allele is expected to cause tumour development .
However, a possibility remains that haploinsufficiency of the normal
TP53 allele could also be responsible for the phenotype . We analysed
two LFS families with nonsense germline TP53 mutations . Nonsensemediated
RNA decay (NMD) eliminates transcripts with premature termination
codons (PTC) . In LFS families with nonsense mutations NMD
could thus influence phenotype, because the removal of mutated transcripts
would lead to the absence of the aberrant protein . TP53 alleles
were analysed in normal tissues (lymphocytes, adrenal gland) and in
tumours of both families on DNA and RNA levels . In normal tissues
the transcripts of mutated alleles were eliminated, similar to some of
the tumours. This may support the notion that TP53 haploinsufficiency,
and not the mutated protein, could be responsible for tumour development
in LFS . However, in some tumours both types of transcripts
(or only mutated transcripts) were present. This could reflect either
differences in NMD between different tissues, or global inactivation of
NMD in tumours . In quest to distinguish between these possibilities
we tested expression of several genes (U2AF35, RPL3, TM1, PTB2,
PTB1, ABCC4) with PTC-containing alternative transcripts, which
should undergo NMD under physiological conditions . However, these
analyses were inconclusive, mainly because the patterns of removal
of some transcripts described in the literature could not be replicated .
Supported by grants MSM0021620813 and MZO00064203 .
P04.156
molecular basis of the Li-Fraumeni syndrome: an update from
the French LFs families
G. Bougeard 1 , R. Sesboüé 1 , S. Baert-Desurmont 1 , S. Vasseur 1 , C. Martin 1 , L.
Brugières 2 , A. Chompret 3 , B. Bressac-de Paillerets 4 , D. Stoppa-Lyonnet 5 , C.
Bonaïti-Pellié 6 , T. Frébourg 1 , &. The French LFS working group 7 ;
1 Inserm U614 and Department of Genetics University hospital, Rouen, France,
2 Department of Pediatric Oncology, Institut Gustave Roussy, Villejuif, France,
3 Department of Medicine, Institut Gustave Roussy, Villejuif, France, 4 Department
of Genetics, Institut Gustave Roussy, Villejuif, France, 5 Department of
Genetics, Institut Curie, Paris, France, 6 Inserm U535, Villejuif, France, 7 -, -,
France.
Extensive analysis of TP53, based on complete sequencing of the 11
exons and QMPSF analysis to detect genomic rearrangements, in
474 French families suggestive of LFS, as defined by the Chompret
criteria, led us to identify in 82 families (17%) a germline alteration
of TP53 . Most of the alterations corresponded to missense mutations
(67%) and we identified in 4 families (5%) genomic deletions. These
results represent a definitive argument demonstrating that LFS results
from TP53 haplo-deficiency. Nevertheless, this does not explain the
remarkable TP53 germline mutation spectrum characterized by the
predominance of missense mutations . The different tumour spectrum
observed between TP53 wt/mt and wt/- mice led us to compare the
mean ages of tumour onset between patients harbouring either TP53
missense mutations or other types of alterations. We found a significant
difference, missense mutations being associated with a 9-year
earlier tumour onset, confirming that missense mutations not only inactivate
p53 but also have an additional oncogenic effect . The observation
of a later age of tumour onset associated with null mutations led
us to perform TP53 analysis in patients suggestive of LFS, but with a
first tumour being diagnosed after 40 years of age and we had the surprise
to identify germline TP53 mutations in such families . Germline alterations
of TP53 that lead exclusively to loss of function are therefore
associated with a later age of tumour onset and the presence of such
mutations should be considered in atypical LFS families with tumours
diagnosed after 40 years .
P04.157
Genetic analysis of tumor cell-free DNA in patients with lung
cancer
E. Hirmerová 1 , A. Panczak 1 , V. Kebrdlová 1 , A. Hořínek 1,2 , A. Slováková 3 , A.
Bortlová 3 , M. Marel 3 , J. Homolka 3 , J. Štekrová 1 , M. Kohoutová 1 ;
1 Inst Biol Med Genetics, 1st Med Fac, Charles Univ, Prague, Czech Republic,
2 3rd Dept Medicine, 1st Med Fac, Charles Univ, Prague, Czech Republic, 3 1st
Dept TBC Resp Dis, 1st Med Fac, Charles Univ, Prague, Czech Republic.
Cell-free DNA (cfDNA) circulating in plasma was found in healthy individuals,
but its concentration can increase in patient with cancer . In
these, total plasmatic cfDNA is the mixture of DNA from normal and
tumor cells . Genetic analysis of tumor cfDNA in plasma could be a
non-invasive tool for early diagnostics and other study of pulmonary tumors
. The concentration of total cfDNA was measured by quantitative
real-time PCR . We analysed total cfDNA by using time-release PCR
with primers for short tandem repeats (STR) located at tumor supressor
genes TP53, APC, and FHIT: diTP53, D5S346, D5S318, D5S299,
D5S82, D3S1300, D3S1560 . We have studied loss of heterozygosity
(LOH) or microsatellite instability by using capillary electrophoresis .
DNA from periferal nucleated blood cells from the same patient served
as a control . We have analysed cfDNA of patients with lung cancer (n =
33), with other pulmonary non-tumor diseases (17), and control group
of healthy individuals (29) . We have proved simultaneous presence of
LOH in multiple STR loci in majority of lung cancer patients in contrast