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

Cancer genetics
lenge for the clinician and for the genetic counselor, who often can not
use them for the management of Lynch syndrome families. Several parameters
can be evaluated to gain insight into the significance of such
unclassified variants (UVs): familial segregation analysis, absence of
the variants in control samples, presence of other pathogenic mutations,
amino acid conservation, and functional and mRNA analyses.
None of these variables can be used alone to predict the significance
of UVs in a single case, but combined evaluation can be clinically useful.
We report our experience on the interpretation of these UVs and
the clinical management of patients and their families.
P0565. Functional screening of MLH1 and MSH2 unclassified
variants in a large cohort of French HNPCC families reveals an
important fraction of splicing mutations
I. Tournier 1 , M. Vezain 1 , A. Martins 1 , F. Charbonnier 1 , S. Baert-Desurmont 1 , J.
Soret 2 , J. Tazi 2 , S. Olschwang 3 , Q. Wang 4 , M. Buisine 5 , T. Frebourg 1 , M. Tosi 1 ;
1 Inserm U614 and Rouen University Hospital, Institute for Biomedical Research,
Rouen, France, 2 IGMM, CNRS UMR 5535, Montpellier, France, 3 Inserm
UMR 599, Institut Paoli-Calmettes, Marseille, France, 4 Molecular Oncology
Unit, Centre Léon Bérard, Lyon, France, 5 Laboratory of Biochemistry and Molecular
Biology, Lille University Hospital, Lille, France.
Numerous unclassified variants (UVs) are detected in the mismatch
repair genes MLH1 and MSH2 involved in hereditary nonpolyposis
colorectal cancer (HNPCC) and cannot be used for genetic counselling.
We have undertaken their systematic screening by developing a
functional splicing assay using patient DNA. The genomic region of
interest, either mutant or wild-type, was PCR-amplified and inserted
into the intron of an expression vector. After transfection into HeLa
cells and total RNA extraction, the effects of mutations on splicing were
evaluated by RT-PCR and systematic sequencing. We have examined
85 different UVs detected in 84 French HNPCC families (54 missense,
10 silent, 3 deletions of a single codon and 18 intronic variants) and
found that 26% affect splicing. Five variants are at positions distinct
from splice sites, suggesting the presence of regulatory elements such
as exonic or intronic splicing enhancers (ESE, ISE). We cloned the
short regions (~30 bp) containing the putative exonic regulatory elements
into the strictly ESE-dependent central exon of a modified βglobin
expression vector and demonstrated by cell transfection assays
and RT-PCR that these sequences indeed contain splicing enhancers.
This led us to examine in this ESE-dependent splicing assay other
MLH1 and MSH2 exonic mutations detected in HNPCC patients and
previously described as affecting splicing and found that the regions
affected by these mutations do contain ESE elements. In absence
of reliable bioinformatics predictions, the sequential use of these two
functional assays represents a valuable tool for the interpretation of
UVs found in these and other genes.
P0566. The significance of BCL-2 and IAP families genes
expression in diagnostics of human acute leukemias
J. Kocki, M. Cioch, A. Dmoszyńska;
Medical University, Lublin, Poland.
The aims of work: (1) The expression levels designation of the genes
regulating the apoptosis process IAP and BCL-2 families genes in the
normal bone marrow cells and the human acute leukaemias cells. (2)
The evaluation of the reciprocal correlations of IAP and BCL-2 families
genes expression, typical for individual types of leukaemias. (3) The
analysis of the potential dependence between the genetic investigations
results and the clinical and laboratory parameters.Conclusions:
(1) The gene expression levels of the IAP and BCL-2 families in normal
bone marrow cells and the human acute leukaemias cells differed in
the individual types of acute leukaemias and had prognostic significance.
(2) In ALL the MCL-1 gene showed the highest expression. (3)
In M0/1 AML the BCL-xL gene showed the highest expression. No dependence
between gene expression levels and the survival, however
the correlations between the BAX or BCL-w genes expression with
CD34 antigen expression were affirmed; (4) In M2 AML the MCL-1
gene showed the highest expression. The dependence of the SUR-
VIVIN gene expression higher level on the bad prognosis for a patient
was affirmed. (5) In M3 AML the MCL-1 gene showed the highest expression.
The dependence of the SURWIWIN, NAIP or MCL-1 genes
expression higher levels on the poor prognosis for a patient were
affirmed. (6) In M4 AML the NAIP gene showed the highest expression.
(7) In M5 AML the expression top-level for the MCL-1 gene was
1 0
affirmed. Supported by grant of Polish State Committee of Scientific
Research No 2 PO5B 064 30.
P0567. I171V mutation in the NBS1 gene is associated with
predisposition to cancer.
J. Nowak1 , D. Januszkiewicz1,2 , M. Mosor1 , I. Ziółkowska1 , K. Rożnowski3 , M.
Wierzbicka3 ;
1Institute of Human Genetics Polish Academy of Sciences, Poznań, Poland,
2 3 University of Medical Sciences, Poznan, Poland, University of Medical Sciences,
Poznań, Poland.
Homozygous mutation 657del5 within the NBS1 gene is responsible
for the majority of Nijmegen breakage syndrome (NBS) cases. NBS
patients are characterized by increased susceptibility to malignancies
mainly of lymphoid origin. Recently it has been postulated that heterozygous
carries of 657del5 NBS1 mutation are at higher risk for cancer
development. The aim of the study was to analyse the frequency of
I171V mutation in NBS1 gene in 135 children with acute lymhoblastic
leukemia, 258 women with breast cancer, 176 patients with larynx
cancer, 103 with second primary tumours and 385 healthy individuals.
I171V mutation was present in 22 cancer patients compared with only
one in healthy individuals. This constitutes 3.27% in studied patients
with malignancies and 0.26% in the control group (P=0.0006; relative
risk, 0.657; odds ratio, 0.077; 95% confidence interval, 0.01-0.57).
Since DNA was isolated from non malignant cells, all mutations found
in cancer patients appeared to be of germinal origin. It can be concluded
that NBS1 allele I171V may be a general cancer susceptibility
gene.
P0568. Intracranial germ cell tumors: association with Klinefelter
Syndrome and analysis of X/Y chromosome aneuploidies in the
tumors
S. Kofman-Alfaro 1,2 , K. Nieto 1,2 , D. Aguirre 1 , I. Palma 1 , R. Peña 3 , G. Queipo 1,2 ;
1 Hospital General de Mexico, Mexico City, Mexico, 2 Facultad de Medicina Universidad
Nacional Autónoma de México, Mexico City, Mexico, 3 Hospital Infantil
de México Federico Gómez, Mexico City, Mexico.
Extragonadal germ cell tumors (EGCT) arise in specific midline regions
mainly in the anterior mediastinum, intracranial germ cell tumors
(IGCT) occur mainly in male children and adolescents. X chromosome
polyploidy and X hypometilation have been suggested as a mechanism
for malignant transformation independently of the histological type. On
the other hand, several reports associated EGCT with Klinefelter syndrome
(KS). Recent reports indicate that KS patients have a relative
risk (66.7) for development of malignant mediastinal germ-cell tumors
and around 8% of male patients with primary mediastinal tumors have
KS, corresponding to 50 times of the expected frequency. Trying to
record the frequency of KS and to confirm the presence of X chromosome
polyploidy in XY IGCT, 13 paraffin embedded tumoral and normal
tissue specimens in XY cases with IGCT were studied using FISH.
We confirm KS in two cases (8%) demonstrating that this constitutive
aneuploidy could be related to carcinogenesis. A low percentage (1%)
of X and Y chromosome polyploidy was observed in all cases. In the
XY patients the aneuploidy could be involved in the tumorogenesis,
however, malignant transformation arise through the accumulation of
multiple and different genetic abnormalities.
P0569. Inactivation of the laminin gamma 3 chain (LAMC ) gene
in various cancers
E. B. Kuznetsova 1,2 , D. S. Mikhaylenko 1,2 , E. A. Pudova 3 , S. S. Larin 4 , M. V.
Nemtsova 1,2 , D. V. Zaletayev 1,2 , V. V. Strelnikov 1,2 ;
1 Institute of Molecular Medicine, Moscow, Russian Federation, 2 Research Centre
for Medical Genetics, Moscow, Russian Federation, 3 Russian State Medical
University, Moscow, Russian Federation, 4 Institute of Gene Biology, Moscow,
Russian Federation.
We have identified LAMC3 gene promoter CpG island among those
abnormally methylated in breast cancer by methylation sensitive arbitrarily
primed PCR. Gene expression analysis by real-time RT-PCR
has revealed extremely frequent (94%) decrease/loss of LAMC3 expression
in breast cancer samples. Loss of expression was also detected
in the MCF7 breast cancer cell line. We have performed the
fine mapping of the CpG island and designed a PCR assay for LAMC3
methylation detection. Its promoter region appeared to be unmethylated
in control samples and methylated in several breast cancer samples,
but the frequency of this abnormal methylation was surprisingly