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

Cancer genetics
P0542. Differential methylation of HLAIII locus (6q21) in normal
and cancer cervical tissues
D. V. Gra 1 , T. L. Azhikina 2 , N. P. Kiseleva 1 ;
1 Institute of Carcinogenesis, N.N. Blokhin Cancer Research Center of Rus.
Acad. Med. Sci., Moscow, Russian Federation, 2 Shemyakin and Ovchinnikov
Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow,
Russian Federation.
Epigenetic alterations, such as abnormal DNA-methylation patterns,
are associated with many human tumor types. New techniques have
been developed to perform genome-wide screening for alterations in
DNA-methylation patterns to identify new tumor-suppressor genes and
to find patterns that can be used in diagnosis and prognosis.
Non-methylated Genomic Sites Coincidence Cloning (NGSCC) allows
to analyze tissue-specific distribution of the unmethylated CpG
sites within the megabase(s)-long genomic DNA fragments (NGSCC;
Azhikina et al., Mol Gen Genomics, 271: 22-32). We applied this technique
to the HLA III locus (6q21), containing D6S273, the known as
a marker of early genetic alterations in cervical neoplasias. Heterozygocity
loss in this specific locus correlates with tumor progression
and suggests that there are potential tumor-suppressor genes in this
region of the chromosome 6 (Mazurenko et al., Mol Biol (Mosk), 40:
436-447).
With the use of NGSCC we constructed high density maps of unmethylated
CpGs for both cancer and normal cervical tissues. We found
the methylation patterns of CpG’s in promoter region of BAT2, BAT3
and MICB differ in normal and cancer tissues. These results were confirmed
by bisulfite sequencing of the CpG islands of these genes.
Thus, the applied approach revealed characteristic tissue-specific features
of large-scale distribution of unmethylated CpGs. The changes
observed in this distribution might provide useful epigenetic markers of
cancerous transformation.
P0543. Studying of adenovirus E1A gene silencing effects on
HEK 293 cancerous cells using RNAi technique
H. Vosgha, M. Behmanesh, M. Sadeghizadeh;
Tarbiat Modares University, Tehran, Islamic Republic of Iran.
RNA interference (RNAi) is a natural gene regulatory mechanism
widely used for studying gene function in a variety of species. Small
interference RNA (siRNA) technology has been reported to produce
post-transcriptional gene silencing (PTGS) in mammalian cells. Studies
have been shown that siRNA expression mediated by vectors
causes efficient and stable down regulation of gene expression, resulting
in functional inactivation of the targeted genes. So in this study, we
designed a human U6 promoter-driven mammalian expression vector
to produce small hairpin RNA (shRNA) for E1A gene transcripts. To
transfect HEK 293 cells by our designed plasmid, dendrosome, a new
designed chemical compound was used. Using this technique, we got
a system for stable expression of shRNA to reduce Ad5 E1A gene transcripts
in these cells. In this report we will present the result of E1A silencing
effects on cell cycle genes such as Rb1 by gene expression.
P0544. Mutational analysis of the beta-catenin gene in MSI(+)
and MSI(-) endometrial tumors from bulgarian patients
D. V. Konstantinova 1,2 , T. K. Kadiyska 1 , R. P. Kaneva 1 , S. I. Ivanov 3 , R. G.
Dimitrov 4 , T. V. Dyankova 3 , E. Tiufektchieva 5 , K. P. Meinhardt 6 , N. I. Doganov 7 ,
V. I. Mitev 2 , I. M. Kremensky 1 ;
1 Laboratory of Molecular Pathology, University Hospital of Obstetrics and Gynecology
“Maichin Dom”, Sofia, Bulgaria, 2 Department of Chemistry and Biochemistry,
Medical University, Sofia, Bulgaria, 3 Clinic of Oncogynecology, National
Centre of Oncology, Sofia, Bulgaria, 4 II Clinic of Operative Gynecology, University
Hospital of Obstetrics and Gynecology “Maichin Dom”, Sofia, Bulgaria, 5 I
Clinic of Operative Gynecology, University Hospital of Obstetrics and Gynecology
“Maichin Dom”, Sofia, Sofia, Bulgaria, 6 Department of Pathology, University
Hospital “Aleksandrovska”, Sofia, Bulgaria, 7 University Hospital of Obstetrics
and Gynecology “Maichin Dom”, Sofia, Bulgaria.
Activating somatic mutations in exon 3 of the ß-catenin gene (CTN-
NB1) have been identified in 11 - 25% of endometrial cancer patients.
Attempts to systematize their distribution among microsatellite unstable
MSI(+) and stable MSI(-) tumors have produced conflicting results.
Tumor MSI has been reported at frequences from 17 to 45% for endometrial
cancer.
In order to evaluate the frequency of exon 3 CTNNB1 mutations and
their possible association with MSI status, we studied 35 patients with
histologically confirmed stage I/II endometrial cancer.
DNA was extracted form fresh tumor tissue and from whole blood. For
MSI status determination we used a panel of six polymorphic markers
- BAT26, D2S123, D5S346, D18S35, FGA, and TP53. MSI(+) were
defined cases where two or more markers showed instability. CTNNB1
exon 3 was direcly sequenced.
We detected MSI in 10 tumors (28.6%). Two of them (20%) harbored
CTNNB1 mutations - a transversion at Ser 37 and a previously undescribed
deletion of 48 bp corresponding to loss of codons 31 to 47.
MSI(-) tumors had no mutations.
As a downstream transcriptional activator in the Wnt pathway, ßcatenin
is regulated by phosphorylation on Ser/Thr sites encoded by
exon 3 of the gene. Mutations at these residues often lead to nuclear
accumulation of the protein and activation of target genes c-Myc and
cyclin D1.
We identified mutations that alter or abolish Ser/Thr residues implicated
in the down-regulation of ß-catenin. In our patient group their
frequency is 5,7% and they are associated with MSI positive cases
(p=0.021).
P0545. Identification of DNA methylation markers for detection
and classification of colon cancer by epigenetic profiling
E. H. J. van Roon, M. van Puijenbroek, H. Morreau, J. M. Boer;
LUMC, Leiden, The Netherlands.
Promoter methylation is thought to be an initial event in tumorigenesis.
Especially right- sided colon cancer is associated with epigenetic
modification of gene expression. The mismatch repair gene MLH1 is
a prime target for epigenetic downregulation with a frequency of 40%
in all right-sided colon carcinomas. Our aim in this study is to identify
additional epigenetic markers to generate a panel that will allow the
epigenetic detection and classification of colorectal cancer (CRC) and
its precursor forms.
We carried out a genome-wide methylation study on clinically welldescribed
colorectal adenocarcinomas, adenomas and paired normal
epithelium. DNA from macrodissected fresh-frozen tissue was digested
by the restriction enzyme MseI, linker-ligated and subsequently digested
by two methylation-sensitive restriction enzymes. The remaining
fragments were amplified, labeled and hybridized to our 9K clone
library CpG island microarrays. Recently, we extended the screen to
high-density CpG island tiling oligonucleotide microarrays.
We identified several loci aberrantly methylated with a high frequency
in carcinomas using our 9K array. Thusfar three loci were verified using
direct- and clonal bisulfite sequencing and are currently being studied
in more detail. Interestingly, one locus was shown to be hypermethylated
in carcinomas as well as adenomas. Possible clinical applications
of these markers include early detection of cancer with applications
such as in feces screening, and cancer prognosis.
P0546. FISH studies using bac clones of the EVI1 locus
in 9 patients with hematological malignancies carrying 3q
rearrangements.
D. Costa 1 , I. Madrigal 2 , A. Carrió 1 , C. Gómez 1 , M. Rozman 3 , J. Esteve 3 , B.
Nomdedeu 3 , E. Campo 1 ;
1 Hematopathology Unit. Hospital Clínic, Barcelona, Spain, 2 IDIBAPS, Biochemistry
and Molecular Genetics Department, Hospital Clínic. Barcelona, Spain,
3 Hematology Service. Hospital Clínic, Barcelona, Spain.
Chromosomal rearrangements involving 3q26 are a recurrent aberration
in malignant myeloid disorders. Several of these rearrangements
involve the EVI1 oncogene or its surrounding sequences and are associated
with a poor prognosis. In order to know whether the EVI1 locus
was rearranged in 9 patients with hematological malignancies carrying
3q abnormalities, fluorescent in situ hybridization (FISH) studies using
BAC (bacterial artificial chromosome) clones were carried on. A dual
color probe was constructed with 9 BACs; centromeric clones covering
1Mb and including EVI1 gene were labeled with a red fluorescent dye
and telomeric clones covering 1 Mb were labeled with a green fluorescent
dye. From the 9 patients, two patients showed normal copies of
the EVI1 locus, four patients showed one EVI1 locus rearranged and in
all of them the breakpoint on 3q26 was telomeric to EVI1 gene, one patient
showed one copy of the EVI1 locus translocated to another chromosome,
one patient showed one copy of the EVI1 locus rearranged
and the other copy translocated and one patient showed one extra
copy of the EVI1 locus. FISH studies using the EVI1 clones allowed
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