ABSTRACTS
ESHG - Posters: P 2 Cancer Genetics
P0045
Germline Mutations in BRCA1 and BRCA2 genes in the Czech Hereditary Forms of
Breast / Ovarian Cancer
E. Machackova, M. Navratilova, H. Pavlu, D. Valik, M. Hruba, L.
Foretova;
Masaryk Memorial Cancer Institute, Brno, CZECH REPUBLIC.
Background: It is estimated that 5-10% of all breast cancers can be of
hereditary origin. Germline mutations in highly penetrant cancer
susceptibility genes BRCA1 and BRCA2 could cause genetic predisposition to
breast and ovarian cancers.
Material and Methods: Molecular genetic analysis of BRCA1 and BRCA2 genes was
performed in 150 high-risk breast and breast/ovarian cancer families and in 25
women diagnosed with early-onset sporadic breast cancers below 40 years.
Protein truncation test and heteroduplex analysis followed by sequencing was
carried out on genomic DNA isolated from blood samples. The genetic counseling
and preventive clinical follow-up of gene carriers is part of the genetic
program.
Results: A germline disease causing mutation was found in 63 screened
high-risk unrelated families (42%), 41 mutations (12 different) in BRCA1 gene
and 22 mutations (15 different) in BRCA2 gene. The most frequently detected
mutations in BRCA1 gene were 5385-5386insC (14 families) and 3819-3823del5 (7
families). Two novel frame shift mutations were detected in BRCA1gene:
2616-2617ins10; 3761-3762del2. Three novel frame shift mutations were detected
in BRCA2 gene: 5073-5074del2; 6677-6678del2; 6866delC. Within sporadic
early-onset breast cancer cases no disease-causing mutation was found.
Conclusion: Germline mutations in BRCA1 and BRCA2 genes are responsible for a
significant fraction of familial breast and ovarian cancer cases in the Czech
Republic.
Contract grant sponsor: Ministry of Health of the Czech Republic:
MZ00020980501;
and Internal Grant Agency of the Ministry of Health of the Czech Republic:
NC5561-3 and NC6396-3.
P0046
Screening of the BRCA1 and BRCA2 genes in western Sweden
Y. Engwall 1, M. Nordling 1, J. Lundberg 1,
A. Bergman 1, T. Martinsson 1, Z. Einbeigi 2, P.
Karlsson 2, A. Wallgren 2, J. Wahlström 1;
1Dept. of Clinical Genetics, Sahlgrenska Univ. Hospital/East,
Göteborg, SWEDEN, 2Dept. of Oncology, Sahlgrenska Univ.
Hospital/Sahlgrenska, Göteborg, SWEDEN.
Germline mutations in the hereditary breast/ovarian cancer causative genes
BRCA1 and BRCA2 are considered to constitute approximately 6-10% of these
cancers. The frequency of female mutation carriers with breast/ovarian cancer
depends on the population studied, and display considerable variation in
coincidence with ethnic and geographical diversity. Mutations are mainly found
as small insertions, deletions or substitutions, but also as exon-wide
deletions. We performed mutation analyses in 116 patients, selected under
informed consent, from the Sahlgrenska University Hospital, Gothenburg,
Sweden. The genes were initially screened using the Protein Truncation Test
(PTT) on genomic DNA (BRCA1 exon 11, BRCA2 exon 10, 11) and cDNA from RT-PCR
(all other exons) for truncating mutations. All mutations but one was detected
with PTT; the remaining one with dHPLC. Automated DNA sequencing of the
detected mutations revealed seven different frameshift mutations, two nonsense
mutations and one large deletion. Four of these have not been reported
earlier: BRCA1 409-410delCA; 2229-2230delAA; 3029delA; 1912 T>G. BRCA
mutations were found in 34% of the screened.families; this is comparable to
frequencies reported in other European studies. Notably, a western Swedish
founder mutation (BRCA1 3171ins5) accounted for 27 of the 37 mutations
detected in the 116 families. Our results are furthermore in accordance with
the observation that frameshift mutations in the first two-thirds of BRCA1 are
associated with a higher risk of ovarian relative to breast cancer than are
truncating mutations in the last one-third of the gene.
P0047
An investigation into the methylation status of oestrogen receptor beta and
its subsequent expression in human breast cancer
F. J. Cooke 1, P. Balfe 1, A. H. McCann 2,
P. Dervan 3, M. Kennedy 3, M. Kerin 1;
1Conway Institute of Biomolecular and Biomedical Research, University
College Dublin / Mater Hospital, Dublin, IRELAND, 2Conway Institute
for Biomolecular and Biomedical Research, University College Dublin, IRELAND, 3Department
of Pathology, Mater Misericordiae Hospital, Eccles St, Dublin 7, IRELAND.
Since the discovery of the novel oestrogen receptor beta (ER
b)
much investigation has centered on the role of this new marker in the
prognosis of breast cancer and response to adjuvant tamoxifen. The aim of this
study is to assess the methylation status of the ER
b
promoter with respect to ER
b expression by
immunohistochemistry in a preliminary cohort of 25 primary human breast
tumors. In addition we sought to correlate ER
a, ER
b
and c-erbB2 status with tumor staging and prognosis.
Fresh tissue was prospectively collected from screen detected and symptomatic
palpable breast tumors managed in the Mater Misericordiae and Mater Private
Hospitals. DNA was extracted from the frozen tissue using the standard
phenol-chloroform approach. The DNA was subsequently quantified by
spectrophotometry and its integrity verified on a 1% agarose gel. To allow
Methylation Specific PCR, the DNA was modified by a sodium bisulphate
procedure and amplified using methylation specific primers. Resultant products
were analysed on an 8% PAGE and confirmed with sequencing.
Table of Contents
Table 1. Histological diagnosis and immunohistochemical status
| Diagnosis |
Total |
ERa
status
(% positive) |
ERb
status
(% positive) |
| Invasive
Ductal |
22 |
20/22
(91%) |
16/22(73%) |
| Invasive
Lobular |
3 |
3/3 (100%) |
2/3(66%) |
With the establishment of this novel MSP approach to analyzing the methylation
status of the ER
b promoter in human breast cancer,
we intend to correlate the findings with the immunohistochemical results
obtained with the 14C8 monoclonal antibody. These studies are of special
importance in the context of epigenetic reversibility as a potential
therapeutic option.
P0048
Analysis of mutations in the BRCA2 gene in Chilean families with breast
cancer
M. Carvallo 1, L. Palma 1, M. Gallardo 1,
C. Rousseau 2, M. King 2;
1Universidad Catolica de Chile, Santiago, CHILE, 2University
of Washington, Seattle, WA.
Two genes have been described until today as responsible for familiar breast
cancer, BRCA1 and BRCA2. Several studies have demonstrated that the frequency
of mutations in either gene is variable depending on the population analysed.
Since this effect may be due to the ethnic origin of the families selected, we
were interested in knowing the incidence of BRCA1 and BRCA2 mutations in
Chilean families. We previously reported a very low frequency of mutations in
the BRCA1 gene (10%). This study shows the analysis of BRCA2 mutations in the
same group of families. The families were selected by standard criteria. All
exons encoding the BRCA2 gene were PCR amplified and analysed through SSCA,
heteroduplex and DNA sequencing. We found in one family the 6174delT mutation
in exon 11, which has been extensively reported in families with
Ashkenazi-Jewish ancestors. The patient carrying the mutation informed about
Ashkenazi-Jewish ancestors in her family. The second mutation found is
6857delAA, also present in exon 11. This is a very interesting mutation for
our study, since it has been described previously in three families from
Spanish origin. Due to high percentage of admixture of the Chilean population
with Spanish colonisers during the XVI and XVII centuries, it is highly
probable that the mutation has a common ancestor. Also it is interesting to
note that the incidence of BRCA1 and BRCA2 mutations in the Spanish population
is below 20% each. (Financed by FONDECYT 1011076)
P0049
Telomerase enzyme activity and chromosome abnormalities detected by combined
G-banding and comparative genomic hybridization in primary breast cancer
A. Papadopoulou 1, T. Trangas 1, H. Tsarouha 1,
M. Texeira 2, E. Dimitriadis 1, P. Ioannidis 1, S.
Heim 3, N. Pandis 1;
1"G. Papanikolaou" Research Center, "Saint
Savvas" Oncological Hospital of Athens, Athens, GREECE, 2Portugese
Oncology Institute, Porto, PORTUGAL, 3The Norwegian Radium Hospital,
Oslo, NORWAY.
Several structural and numerical chromosomal abnormalities have been recorded
in breast cancer. It has been suggested that some chromosomal aberrations may
be the result of telomere dysfunction in rapidly proliferating cells. The de
novo activation of telomerase in cancer possibly provides a survival mechanism
curtailing further chromosomal aberrations. In order to investigate the
relation of telomerase level of expression and the extent of chromosomal
aberrations, 62 primary breast carcinomas were studied. Telomerase activity
was measured using a PCR based TRAP assay and 92% of the tumors were found to
express Telomerase with relative activity ranging from 0 – 3839.6. Genetic
alterations were determined by G banding and Comparative Genomic Hybridization
analysis. 97% of the tumors exhibited chromosomal aberrations ranging from
0-45. The average number of genetic alteration recorded by G-banding and CGH
was 10.98. No correlation was observed between Telomerase activity levels and
the number of genetic alteration in the overall sample population. However
when tumor samples with below average genetic alteration numbers were
considered as a separate group, a statistically significant positive
correlation was recorded between Telomerase activity levels and number of
genetic alterations (R=0.339, p=0.032). This relationship was inversed in the
sample group with above average genetic alterations but it was not
statistically significant (R=-0.127; p=0.574). These results suggest that
Telomerase may be activated in the initial steps of carcinogenesis perhaps to
maintain chromosomal integrity of the rapidly proliferating tumor cells while
at the later stages alternative survival mechanisms have evolved.
P0050
Selective genetic screening in 250 Belgian breast and ovarian cancer families
identifies BRCA1 or BRCA2 mutations in 20% of cases
G. Michils, K. Minner, B. Vankeirsbilck, E. Legius, G. Matthijs;
Center for Human Genetics, Leuven, BELGIUM.
Germline mutations of the BRCA genes are associated with familial breast
and/or ovarium cancer, or with early onset of breast cancer (<35y) in the
absence of a familial history. In families attending a cancer genetic clinic
mutations are typically identified in 15-20% of the analysed families. Most
mutations are truncating and spread all over the coding regions, but at a
higher frequency in the large exons (exon 11 of BRCA1 and exons 10 and 11 of
BRCA2). We chose to analyse these large exons together with exons with Belgian
founder mutations, Ashkenazi mutations, hot spots and exonic deletions.
DNA samples of a cohort of 250 unrelated affected patients were included in
this study: 140 samples were analysed by Enzymatic Mutation Detection (EMD),
while 110 samples were screened by Denaturing High Performance Liquid
Chromatography (DHPLC). Exon deletions were analysed by amplification across
breakpoint junctions.
By screening selected exons of BRCA1 and BRCA2, 21 germline truncating
mutations and one exonic deletion were found in 48 of 250 families. In
addition, one pathogenic missense mutation has been identified in BRCA1: M1V.
DHPLC has advantages in comparison to EMD, mainly because it is
semi-automatable. This study shows that a limited screening of BRCA1 and BRCA2
results is a high yield of mutations in our clinical sample (20%). This
screening could be offered to a large group of females while an exhaustive
screening could be performed in a more selected group. Analysis of additional
exons by DHPLC is going on in such a selected group.
P0051
RAD6 and breast cancer
A. Lyakhovich 1 ,2, M. Shekhar 1;
1Wayne State University, Detroit, MI, 2Institute of
Molecular Biology and Biophysics, Novosibirsk, RUSSIAN FEDERATION.
Treatment with DNA damaging drugs is commonly used to localize breast cancer.
It causes DNA damage and leads to genomic instability and/or apoptosis as a
result of mutation or altered expression of genes associated with DNA repair,
in particular RAD6. RAD6 (Ubch2) protein is present at low amounts in
cytoplasm of normal human breast cells, while in metastatic breast cancer
cells it is up-regulated and localized in the nucleus. We have demonstrated,
that overexpression of exogenous RAD6 cDNA in MCF10A human breast epithelial
cells induced cell-cell fusion generating multinucleated cells, centrosome
amplification, abnormal mitosis and aneuploidy. We found that exposure of
MCF10A cells with cisplatin or adriamycin resulted in enhancement of RAD6
mRNA/protein level which was post-transcriptionally regulated and
post-translationally stabilized. RAD6 protein is predominantly expressed
during late S/G2 phases. Its localization in cells at specific stages of
mitosis reveals the lack of association of RAD6 with condensed chromatin.
Co-localization of RAD6 protein with g-tubulin on
centrosomes is maintained throughout the interphase and mitotic stages of the
cell cycle. We were able to show for the first time that in drug-treated cells
RAD6 is associated with p53-p14ARF-MDM2 in the nucleus. The expression of RAD6
correlates with human breast cancer stage and can be used as a marker to
predict response to chemotherapy. Our findings suggest that RAD6 at low levels
may play a significant role in the maintenance of genomic integrity of
mammalian cells, high levels of RAD6 probably over a certain threshold may
cause genomic instability.
P0052
Elevated CA-125 serum level as an example of correlation between cell
biology, carcinogenesis, positive family story. A case of woman from the
breast/ovarian cancer family, with mutation in BRCA1 gene.
K. Kaczanowska;
Children`s University Hospital, Lublin, POLAND.
Characteristic feature of the neoplasm evolution is long-lasting process. The
priority of oncology is to diagnose cancer in its pre-clinical stage of
development. It is extremely important in families, which have positive family
story. The point is searching for substances, presence of which in the blood
would give evidence to the presence of cancer. In case of CA-125, its
production in tumor is significantly higher than in a normal cell. Level
depends on mass of the living tumor cells.
Here we report a case of patient, whose family was affected by three breast
and one ovarian cancer. Because strong aggregation breast and ovarian cancer-
the woman performed genetic test. It showed mutation in BRCA1 gene, exon
20-5382insC. The next two mutations were proven in her sister daughters.
In all performed clinical examinations (mammography, ultrasonographic
examination of the breasts, transvaginal ultrasonography) there were no
pathology. But the serum level CA-125 was highly increased, accordingly:108
and 125 IU/L ( normal: 30 IU/L). She underwent prophylactic oophrectomy, and
at the time of surgery tumor was suspected. Histopathology gave the final
solution: poorely diffrentiated adenocarcinoma, only in one site of the left
ovary. As a consequence, the woman started few courses of chemotheraphy.
We want to conclude, that we should perform BRCA1 and BRCA2 tests in families
with strong aggregation of breast and ovarian cancer. We should have a high
suspicion of cancer, when serum level of Ca-125 is highly increased. We want
to undreline the strategy of prevention.
P0053
Androgen Receptor CAG Repeat Length in Jewish Israeli Women who are BRCA1/2
Mutation Carriers: Association with Breast/Ovarian Cancer Phenotype
E. Dagan 1, E. Friedman 2, T. Paperna 1,
N. Carmi 2, R. Gershoni-Baruch 1;
1Rambam Medical Center, Haifa, ISRAEL, 2Sheba Medical
Center, Tel-Aviv, ISRAEL.
BRCA1/2 mutation carriers are at increased lifetime risk for developing breast
and/or ovarian cancer. Yet, the genetic or environmental factors that govern
the phenotypic expression of mutant BRCA1/2 alleles remain elusive. The CAG
repeat, within exon 1 of the Androgen Receptor (AR) gene is reportedly
associated with breast cancer phenotype in BRCA1 mutation carriers. To extend
this observation, we genotyped 227 BRCA1/2 mutation carriers for the
polymorphic AR CAG repeat, and correlated allele size with breast/ovarian
cancer morbidity parameters. Of 227 BRCA1/2 carriers, 169 were BRCA1 mutation
carriers and 58 carried a BRCA2 mutation. Seventy-nine women had unilateral
breast cancer, 15 - bilateral breast cancer, 41 - ovarian cancer, 14 - breast
and ovarian cancer and 78 were asymptomatic mutation carriers. Mean age at
diagnosis in women with either or both neoplasms was 46.7±11.2 years, and
that of the asymptomatic group - 45.8±9.4 years, a statistically
insignificant difference. The AR CAG repeat ranged from 8-28 in all tested
women. Mean number of AR CAG repeat was not statistically different between
affected (18.3±2.4) and asymptomatic mutation carriers (18.6±2.1). AR CAG
repeat among patients with early onset (<42 years) breast cancer was
significantly shorter (17.5±2.3) compared with asymptomatic individuals
(18.6±2.1) (p<0.01), and the shorter allele - the younger the age at
diagnosis. This study does not provide conclusive evidence of association
between AR CAG repeat size and breast or ovarian cancer risk. However, a small
effect of a short AR CAG allele size on breast cancer penetrance at early age
was noted.
P0054
Association of 5382insC Mutation with SNPs of BRCA1 Gene and the Mutation
Frequency in Russia
A. N. Loginova 1, N. I. Pospekhova 1, L. N.
Lubchenko 2, E. V. Khomich 1, I. V. Kuzmina 1, A.
V. Budilov 3, V. M. Zakharyev 3, E. K. Ginter 1,
R. F. Garkavtseva 2, A. V. Karpukhin 1;
1Research Centre For Medical Genetics, Moscow, RUSSIAN FEDERATION, 2Cancer
Research Centre, Moscow, RUSSIAN FEDERATION, 3Engelgardt Institute of
Molecular Biology, Moscow, RUSSIAN FEDERATION.
A high predominance of 5382insC in BRCA1 gene mutation spectrum (80% of all
mutations) of patients with familial breast/ovarian cancer and a set of 11
SNPs on an extent of the gene were found. This set of SNPs in strong linkage
disequilibrium and consensus sequence defined two most frequent haplotypes
(named B and A). The haplotype frequencies in cancer patients and in control
individuals were not different significantly. However, the haplotype A to the
haplotype B ratio in genomes with the 5382insC mutation was approximately
three times higher than these haplotypes ratio in the population (P <
0.04). The same difference between patients under 5382insC mutation and
control group was observed for genotype frequencies (P < 0.04) with odds
ratio equal 3.3 in favor of AA among patients. The reason for observed
frequency difference may be the mutation - haplotype A linkage. But it is
interesting that a ratio of genotype AA and AB frequencies under the mutation
is different in patients and control individuals. This may suggest on
operation of other factors in addition to linkage. It should be noted that the
haplotypes A and B frequencies in Russian and West-European patients with
5382insC were the same (P = 0.40).
The frequency of 5382insC mutation revealed in Russia is highest among
investigated populations. The proportion of this mutation is next highest in
East-European countries and common in West-Europe. These data jointly with the
same SNP haplotypes found in Russia and West-Europe are suggestive on
East-European origin of 5382insC mutation.
P0055
BRCA2 mutations and polymorphisms in Russian patients with familial
breast/ovarian cancer
E. V. Khomich 1, N. I. Pospekhova 1, L. N.
Lubchenko 2, A. N. Loginova 1, I. V. Kuzmina 1, A.
V. Budilov 3, V. M. Zakharyev 3, E. K. Ginter 1,
R. F. Garkavtseva 2, A. V. Karpukhin 1;
1Research Centre for Medical Genetics, Moscow, RUSSIAN FEDERATION, 2Cancer
Research Centre, Moscow, RUSSIAN FEDERATION, 3Engelgardt Institute of
Molecular Biology, Moscow, RUSSIAN FEDERATION.
BRCA1 mutations were found in 40% of a sample of breast/ovarian cancer
families in Russia. In present study BRCA2 gene sequence variations among
cancer families of the rest part of the sample were investigated.
There were only 11% of the probands with deleterious BRCA2 mutations. Two
deleterious mutations - 2001del4 and 4816insG - are new. Missence mutations of
unclear significance were revealed in 19% of the cases. One of that (N1808K)
and two single nucleotide polymorphisms (SNPs) are revealed for the first
time. The gene variant S384F that was thought to be unclear significance
mutation is evidently polymorphism because was found in common with
deleterious mutation of BRCA2 gene. SNPs of 12 types on an extent of BRCA2
gene were found. Six of those were in coding regions of the gene. A variant
N372H that known as confers an increased breast cancer risk under HH
homozygote, in 12% of the cases was homozygosis on HH with allele frequency
equals 0.31. At the same time, a variant IVS11+80del4 with the similar allele
frequency was not found as homozygote. It is interesting that we found a
frequency of 203G/A polymorphism significantly higher in comparison with
results in BIC data base, although the frequencies of other frequent SNPs were
not so different. BRCA2 SNPs of control group are under investigation at
present.
P0056
Prevalence of BRCA1 gene 5382insC mutation in St.Petersburg patients with
familial breast cancer.
N. A. Grudinina 1, E. P. Lamber 2;
1Insitute for Experimental Medicine, Saint Petersburg, RUSSIAN
FEDERATION, 2Institute for Experimental Medicine, Saint Petersburg,
RUSSIAN FEDERATION.
The BRCA1 gene mutation are the common cause of familial breast cancer. The
risk of breast cancer development in women with germline BRCA1 gene mutations
approaches 90% during life span. We have created the DNA collection from St.
Petersburg familial breast cancer patients and demonstrated nearly the same
frequency of BRCA1 gene 5382insC mutation in both Slavic and Ashkenazi Jewish
patients with familial breast cancer. 5382insC mutation of the BRCA1 gene was
found in 1 Ashkenazi Jewish family with familial breast cancer out of 9
studied and in 3 Slavic families with familial breast cancer out of 20
studied. 5382insC mutation was found neither in Ashkenazi and Slavic patients
with sporadic breast cancer, nor in control group, that consists of 50 Slavic
and 50 healthy Ashkenazi patients unselected in respect of familial breast
cancer. Previously 5382insC mutation of the BRCA1 gene was reported in number
of familial ovary cancer patients from Moscow and thus 5382insC mutation of
the BRCA1 gene may be the common cause of familial breast and ovary cancer in
whole Russia. However, the mutation spectra specificity from other countries
in BRCA1 gene is expected in Russian population and some new BRCA1 gene
mutations are in process of characterization now. The elucidation of BRCA1
gene mutation spectra in St. Petersburg familial breast cancer will help to
provide genetic counseling in breast cancer families and improve treatment
patients with high risk of breast cancer in the future. The current research
was supported in part by RFBR grant 01-04-49627.
P0057
Altered expression of the candidate tumor suppressor gene, WWOX, in human
breast tumors
K. Driouch 1, H. Prydz 2, R. Lidereau 1, E.
Frengen 2;
1INSERM E0017/Oncogénétique, Centre René Huguenin, F-92211
St-Cloud, FRANCE, 2Biotechnology Centre of Oslo, University of Oslo,
N-0316 Oslo, NORWAY.
The presence of putative tumor-suppressor genes on chromosome 16q23.2-24.1 has
been suggested by LOH analysis in breast cancer as well as other cancer types.
This region overlaps with the fragile site FRA16D and the region of homozygous
deletions found in various cancers. We have previously constructed a 1.2 Mb
contig map and used this resource to assign transcripts to the LOH region.
This resulted in the identification of the WWOX/FOR gene.
The mouse homologue of the WWOX protein has been defined as an apoptogenic
protein and an essential partner of p53 in cell death. Thus WWOX is a strong
candidate tumor-suppressor gene. We have performed an expression study of the
WWOX gene in a series of human breast tumors and breast cancer cell lines, and
detected altered WWOX expression at high frequency in cancer cells.
Furthermore, identification of two distinct alternative WWOX transcripts
expressed at high levels in human tumors suggests an involvement of the WWOX
gene in cancer progression. We have initiated functional studies of WWOX in
human cells in order to characterize the role of the WWOX protein in normal as
well as cancerous cells.
This work was supported by the Research Council of Norway, the Norwegian
Cancer Society, the Ligue Nationale de Lutte Contre le Cancer (LNCC), and the
Association pour la Recherche sur le Cancer (ARC).
P0058
Investigation of APC mutations of a patient with FAP and her family members
by heterodublex analyses
B. Tunca 1, M. Menigatti 2, P. Benatti 2,
G. Cecener 1, M. Pedroni 2, A. Scarselli 2, F.
Borghi 2, E. Sala 2, T. Yýlmazlar 3, A. Zorluoglu 3,
U. Egeli 1, O. Yerci 4, M. Ponz de Leon 2;
1University of Uludag, Medical Faculty, Department of Medical Biology
and Genetics, Bursa, TURKEY, 2Dipartimento di Medicina Interna,
Universita di Modena, Modena, ITALY, 3University of Uludag, Medical
Faculty, Department of General Surgery, Bursa, TURKEY, 4University of
Uludag, Medical Faculty, Department of Pathology, Bursa, TURKEY.
Familial adenomatous polyposis coli (FAP) is an autosomal dominant disease
characterised by the presence of 100 or more colorectal adenomatous polyps.
Mutations in the adenomatous polyposis gene (APC) gene primarily responsible
for the development of this disease.
In this study, we examined one patient with FAP and 21 family members
including one effected person from FAP and 20 nonsemptomatic persons. Our
proband case who have a retinal lesions (congenital hypertrophy of the retinal
pigment epithelium, called CHRPE) and hundreds adenomatous polyps on all colon
and rectum is a 36 years old woman. We isolated DNA from pheripheral blood
samples of proband and her family members by proteinaz K incubation and
phenol-chloroform extraction. We studied E,D, F, and G segments of exon 15 of
APC gene by heterodublex analyses (HDA). For staining, we used non-radioactive
silver staining method. We determined mutation in 5 person from this family in
segment F of exon 15 of APC. Two of them were patients with FAP (one is
ourproband case) and another three persons were non semptomatic family
members. Result of sequencing analysis of these cases, we determined T
deletion at position 3554 causing a frameshift mutation in APC gene.
P0059
Involvement of APC/beta-catenin signalling and E-cadherin in sporadic colon
cancer
T. Cacev 1, R. Spaventi 2, K. Pavelic 1,
S. Kapitanovic 1;
1Rudjer Boskovic Institute, Zagreb, CROATIA, 2Pliva d.d.,
Zagreb, CROATIA.
Activation of APC/beta-catenin signalling pathway by mutation in the APC or
beta-catenin gene contributes to colorectal carcinogenesis. E-cadherin is
involved in control of intercellular adhesion and acts as an invasion
supressor. We examined 60 cases of human sporadic colon cancer and
corresponding normal tissue samples to evaluate the loss of heterozygosity
(LOH) and presence of mutations at the APC, beta-catenin and E-cadherin gene
loci.
DNAs were used for PCR, RFLP, VNTR and LOH analysis. To analyze LOH at the APC
gene loci we used three RFLP intragenic markers (exon 11 RsaI, exon 15 MspI,
and exon 15 AspHI). The presence of the mutations in the amplicon 15H of the
APC gene, and APC gene mutation in codon 1309 were analyzed as well. To
analyze mutations in the beta-catenin gene we amplified exon 3 and the
intronic sequences flanking it from tumor DNAs. For the LOH analysis of
E-cadherin gene locus we used D16S752 polymorphic marker.
The informativity for all three APC intragenic markers was 53.3 % (32 of 60
assayed), and 25 % of tumors (8 of 32 informative) demonstrated LOH. We found
two APC gene mutations in our tumor samples: a deletion in codon 1309, and an
insertion in the amplicon 15H of the APC gene. In 3.3 % of tumor samples (2 of
60 tested) the mutation of the beta-catenin gene was found. The informativity
of D16S752 E-cadherin gene polymorphic marker was 75% (45 of 60 tested) and
28.8 % of tumors (13 of 45 informative) demonstrated LOH.
P0060
Genetic analysis of APC gene and the diagnostics of familial adenomatous
polyposis in pediatrics
H. Kapitanovic Vidak, T. Cacev, K. Pavelic, S. Kapitanovic;
Rudjer Boskovic Institute, Zagreb, CROATIA.
Familial adenomatous polyposis (FAP) is an autosomal dominant inherited
disease. Patients with FAP develop hundreds to thousands of adenomatous polyps
in the colon and rectum during their second or third decades and one or more
of them can progress to cancer. Children of affected individuals are at 50%
risk of inheriting the disease. Because FAP patients have a very high risk of
colorectal cancer, identification of the individual risk in family members is
important to prevent cancer deaths. For these at risk members of the family,
annual endoscopy is recommended. The method of providing such accurate
presymptomatic diagnosis is to determine whether a family member has inherited
the particular germ-line mutation of the adenomatous polyposis coli (APC) gene
carried by the affected parent.
Genomic DNAs were isolated from peripheral blood of patients and their
relatives. Polymerase chain reaction (PCR) was performed using specific pairs
of primers. PCR products were analyzed by electrophoresis on a Spreadex EL 300
gels.
The genetic analysis confirmed the APC gene codon 1309 germ-line mutation in
two children not yet having colorectal adenomas, but having inherited APC gene
mutation from their mother who died from colon carcinoma. APC gene mutation
analysis also confirmed the diagnosis of FAP in one child having colorectal
adenomas as a first case of FAP in that family.
We use APC gene mutations analysis in presymptomatic diagnostics but also to
confirm the diagnosis of FAP. Children confirmed as a gene mutation carriers
can be early included in surveillance program and treatment.
P0061
Germline mutations of the APC gene in Czech FAP families
M. Kohoutová 1, J. Stekrová 1, V. Jirásek 2,
J. Kotlas 1, V. Kebrdlová 1;
1Institute of Biology and Medical Genetics of the First Faculty of
Medicine and General Teaching Hospital, Charles University, Prague, CZECH
REPUBLIC, 21st Medical Department of the First Faculty of Medicine
and General Teaching Hospital, Charles University, Prague, CZECH REPUBLIC.
Familial adenomatous polyposis (FAP) is an autosomal dominantly inherited
disease characterised by the development of hundreds to thousands of
colorectal adenomatous polyps and by the progression to carcinomas. Causative
germline mutations have been described in the adenomatous polyposis coli (APC)
gene. In the present study the entire APC coding region has been screened for
germline mutations in 45 unrelated Czech FAP families. Using PCR, DGGE
analysis and DNA sequencing we found 30 mutations, twelve of which were found
to be novel: seven frameshift mutations (exon 9 and 15), two nonsense
mutations (exon 9 and 11) and three splicing mutations (intron 11 and 14). In
previously reported mutations we identified ten frameshift mutations in exon
15 and six nonsense mutations (exon 5, 9 and 15). The common 5 bp deletions at
codons 1309 and 1061 were identified in five families (16,6%) and substitution
2805C>A was found in three cases (10%). Identified mutations result in the
classical form of FAP except the 1 bp deletion at codon 409 (exon 9), which
caused the attenuated form of FAP. In addition, one missense mutation was
revealed in exon 3 although missense mutations are rarely observed in FAP. It
remains to be determined whether this substitution represents the
disease-causing mutation. Two samesense variations were detected in exon 15.
The presence of one of them correlates with disease occurrence in the affected
family. Thus the clinical significance of this mutation cannot be excluded.
Supported by the grant project MS CR:CEZ:J13/98:111100004.
P0062
Denaturing High-Performance Liquid Chromatographie (DHPLC) in Screening for
Mutations in Exon 15 of the APC (Adenomatous Polyposis Coli) Gene
W. Heinritz, A. Kujat, S. Strenge, K. Peisker, U. G. Froster;
University of Leipzig, Department of Human Genetics, Leipzig, GERMANY.
FAP (OMIM: *175100, McKusick 1986) is a rare form of hereditary colorectal
cancer. Germline mutations of the APC gene were reported in patients with
Familial Adenomatous Polyposis (FAP). Inactivation of the APC gene plays a
significant role in the development of early onset colon cancer based on
polyposis of the colorectum. The location of germline mutations in the APC
gene appears to correlate with the clinical phenotype (number of colorectal
adenomas, concomitants like occurence of further adenomas in other digestive
organs, desmoid tumors and Congenital Hypertrophy of the Retinal Pigmental
Epithel [CHRPE]). To provide a fast mutation screening we analyzed the region
of the APC gene where more than 40% of the mutations in FAP are described
(exon 15-4 to 15-8). We established DHPLC (Denaturing High Performance Liquid
Chromatography) mutation analysis followed by automated sequencing of
suspicious fragments. We investigated 9 patients with a clinical diagnosis of
FAP. Three sequence variations could be identified: 1 polymorphism and 2
mutations (3597del2A at codon 1199 with termination of protein translation at
amino acid position 1206; 3949GŕC at codon 1317, E1317Q). We describe the
optimized conditions for DHPLC for this gene. According to our results DHPLC
is an efficient and fast screening method to identify mutations in the APC
gene which can be applied to the other exons of the APC gene for a fast and
cost reducing mutation screening. The rapid mutation screening will optimize
further diagnostic and therapeutic strategies in families with hereditary
colon cancer.
P0063
NF1 tumor suppressor gene in sporadic colon cancer
S. Kapitanovic 1, T. Cacev 1, K. Pavelic 1,
R. Spaventi 1 ,2;
1Rudjer Boskovic Institute, Zagreb, CROATIA, 2PLIVA d.d.,
Zagreb, CROATIA.
Colorectal carcinomas are characterized by multiple genetic alterations that
occur during tumorigenesis. Several tumor suppressor genes associated with
colorectal carcinoma have been identified: MCC and APC on chromosome 5q, p53
on chromosome 17p, nm23-H1 on chromosome 17q, and DCC and DPC4 on chromosome
18q. We examined 60 cases of human sporadic colon cancer and corresponding
normal tissue samples to evaluate the loss of heterozygosity (LOH) at the NF1
gene loci. The purpose of this study was also to evaluate whether the LOH at
the NF1 gene is associated with clinicopathological characteristics in
sporadic colon cancer.
DNAs were used for PCR, RFLP, VNTR, and LOH analysis. PCR was performed using
specific pairs of primers. PCR products were analyzed by RFLP analysis, and
VNTR analysis. To analyze LOH at the NF1 gene loci we used three polymorphic
markers: one RFLP marker (exon 5 RsaI) and two VNTR markers (IVS27AAAT2.1 and
IVS38GT53.0).
Using these three polymorphic markers 50 (83.3%) patients were found
heterozygous and informative for LOH analysis. DNA from 9 (18%) tumors
exhibited LOH at the NF1 locus. The majority NF1 gene LOH was observed in
Dukes' A (56%), in the well differentiated tumors (43%), and in the tumors
that were smaller than 5cm (67%).
Conclusion: Our results support the view that malignant progression is a
consequence of more than one genetic change and suggest that inactivation of
NF1 gene plays a role in a multistep process of colon tumor progression as an
early event.
P0064
Complete characterization of the colon cancer cell line HT29 clone 19A by
multicolor banding (MCB)
A. Kuechler 1 ,2, A. Weise 1, S. Michel 1,
B. Pool-Zobel 3, A. Schaeferhenrich 3, A. Heller 1,
H. Starke 1, T. G. Wendt 2, U. Claussen 1, T.
Liehr 1;
1Institute of Human Genetics and Anthropology, Jena, GERMANY, 2Department
of Radiotherapy, Jena, GERMANY, 3Department of Nutritional
Toxicology, Institute of Nutrition, Jena, GERMANY.
The human colorectal adenocarcinoma cell line HT29 subclone 19A was recently
characterized by M-FISH (Eur J Hum Genet 2001, Vol 9/S1, p138, P0193) and the
following composite karyotype was established:
64~69,XX,+del(Xp),+1,+der(1)t(1;11;16),+2,+der(2)t(1;2),+der(3)ins(3;12),+der(4)t(2;4),+5,+del(5q),+7,+7,-8,+dup(8),+del(9),+der(9)t(6;9;X;9),+10,+11,+11,+del(11p),+del(11q),+12,-13,-13,+i(13q),+i(13q),+der(13)t(5;i(13q)),+15,+16,+17,+del(18),-19,+del(19),+der(19)t(5;19),+der(19)t(17;19),+20,+20,+22,+22[cp10].
Using M-FISH, it was possible to identify the chromosomes involved in
aberrations, but not to define their exact breakpoints. In order to further
clarify the translocation breakpoints and to characterize possible
iso-chromosomes, multicolor banding (MCB) was applied at the 400 band level
according to Mrasek et al. (Cytogenet Cell Genet 93:242-248). MCB-analyses
were performed on all aberrant chromosomes of this composite karyotype, i.e.
on the following eighteen chromosomes: #1, #2, #3, #4, #5, #6, #8, #9, #11,
#12, #13, #16, #17, #18, #19, #20, #22 and X. Where necessary for exact
definition of rearrangements, MCB-probes were combined with centromere
specific and whole chromosome painting probes. The resulting karyotype is as
follows:
64~69,XX,+del(X)(p11.2-->qter),+del(1)(p35-->qter),+2,+der(2)t(1;2)(1q32-->1qter;2pter-->2q11),-3,+i(3)(q10),+der(3)ins(3;12)(3pter-->3p12::12p12::3p12-->3qter),+der(4)t(2;4)(2q35-->2qter;4pter-->4q11),+5,+del(5)(pter-->q11.2),+dic(6;9)t(6;9;X;9)(6pter-->6q10;9q10-->9q21;Xp21.1-->Xp11.3;9q21-->9qter),+7,+7,-8,+dup(8)(qter-->q10::q10-->q24::hsr::q24-->qter),+10,+11,+del(11)(p13-->qter),+der(11)t(11;?)(11pter-->11q24;?),+12,-13,-13,+i(13q),+i(13q),+der(13)t(5;13)(13qter-->13q10::13q10-->13q21::5q31-->5qter),+15,+del(16)(pter-->q13),+der(17)t(19;17)(19pter-->19p11;17p11-->17qter),+i(18p),-19,+der(19)t(5;19)(5pter-->5p11;19q10-->19qter),+20,+20,+22,+der(22)t(17;22;17)
[cp10].
In summary, the MCB-technique was suitable to define all translocation
breakpoints apart from one (i.e. der(22)t(17;22;17) which consists of only
very little chromosomal material). Thus, MCB is a very useful tool for
detailed analyses of chromosomal rearrangements.
Supported by DFG (PO284/6-1), Wilhelm Sander-Stiftung (99.105.1) and EU
(ICA2-CT-2000-10012 and QLRT-1999-31590).
P0065
Optimization of DHPLC experimental conditions for mutation analysis of the
hereditary non polyposis colon cancer (HNPCC) genes hMLH1 and hMSH2
M. Schmitt 1, M. Gribba 2, J. M. Limacher 3,
S. Olschwang 4, J. L. Mandel 2;
1Laboratoire de diagnostic génétique, Strasbourg, FRANCE, 2IGBMC,
Illkirch, FRANCE, 3Service d'Oncologie, Hopital Civil, Strasbourg,
FRANCE, 4CEPH Diagnostics - Laboratoire de Génétique Médicale,
Paris, FRANCE.
Denaturating high-performance liquid chromatography (DHPLC) is an efficient
method for the detection of point mutations in disease-related genes.
Colorectal cancer is one of the most common cancers, and mutations in the
genes for hereditary non polyposis colon cancer (HNPCC), hMLH1 and hMSH2
represent the major cause of hNPCC.
We have recently applied the DHPLC mutation detection to the 16 exons of hMSH2
and 19 exons of hMLH1 genes. To test sensitivity and reproducibility of DHPLC,
we have first determined the best DHPLC conditions on the wild type sequences,
followed by the study of 35 sequence variants previously found by sequencing
DNA samples of HNPCC patients. All of the 35 mutations were detected using
DHPLC (sensitivity 100%).
We then used DHPLC to analyse 18 patients with colorectal cancer not
fulfilling all the Amsterdam criteria. We have found two mutations : Y43C in
exon 1 of hMSH2 (unpublished yet) affecting a highly conserved residue and a
790+1G to A in intron 9 of hMLH1 (previously described), one of them did not
fulfill the Amsterdam criteria. This low mutation yield could be due to the
patients inclusion criteria. We have also found many polymorphisms, with a
much higher frequency than previously published.
In conclusion, DHPLC is a rather rapid and inexpensive technology that may be
used to screen for mutations colorectal cancer patients where HNPCC may be
suspected but who do not fulfill stricter criteria.
P0066
Unusual Findings Of APC Gene Analyses In suspected FAP Cases From The
Republic Of Macedonia
A. J. Dimovski 1, A. M. Stefanovska 1, T.
Josifovski 2, M. Panovski 2, D. Jashar 3, G.
Zografski 3, G. D. Efremov 1;
1Macedonian Academy of Sciences and Arts, RCGEB, Skopje, THE FORMER
YUGOSLAV REPUBLIC OF MACEDONIA, 2Clinic for Abdominal Surgery,
Faculty of Medicine, Skopje, THE FORMER YUGOSLAV REPUBLIC OF MACEDONIA, 3Institute
of Oncology, Faculty of Medicine, Skopje, THE FORMER YUGOSLAV REPUBLIC OF
MACEDONIA.
AIM: Characterization of the molecular basis of FAP in Macedonia.
SUBJECTS: Patients with multiple adenomatous polyposis of the large intestine
confirmed by histopathological evaluation.
METHODS: DGGE of exons 1-14, PTT and heteroduplex analysis of exon 15, RT-PCR
of exons 1-15, sequencing of the 5' end, and Southern blot analysis of the APC
gene.
RESULTS: Six unrelated cases (one female and five males) with multiple
polyposis of the colon were enrolled in this study. Of the six patients, only
one had a positive familial history. In the female patient the number of
polyps was much lower (<100) than the number observed in the male subjects
(>1,000). Detailed DNA analyses of the APC gene revealed the presence of
rare (unusual) defects in two patients. A large deletion, removing the entire
APC gene, was found in the patient with a positive familial history. A somatic
mosaicism for a deletion removing exons 2-14 was detected in the female
patient. No abnormalities at the DNA level and no allelic imbalance in the
expression profile of the APC gene were detected in the other four patients.
Abnormal APC gene transcripts were found in the peripheral blood of two of
these (deletion of exons 9-13 and exon 14, respectively) that could not be
explained by any defect at the DNA level.
CONCLUSION: The unusual findings of our study indicate that there are genes
other than the APC which might influence the development of multiple
colorectal adenomas, either through an APC related mechanism or through other
pathways.
P0067
Linkage mapping of FAP disease modifier locus in a large family with a known APC
mutation
M. Plasilova, Z. Dobbie, K. Heinimann, H. Müller;
Human Genetics, Division of Medical Genetics, University Clinics Basel,
SWITZERLAND.
Familial adenomatous polyposis (FAP) is an autosomal dominant colorectal
cancer predisposition syndrome caused by germline mutations within the
adenomatous polyposis coli (APC) gene. Mouse model studies and broad
phenotypic variability observed both within and among affected families
indicate, that in FAP disease expression also other genetic and environmental
factors must play an important role. Their identification would substantially
improve possibilities of genetic counseling of FAP patients by enabling the
precise prediction of the disease severity. A large FAP kindred which has been
previously reported by our group and harbours an adenine deletion at codon
1982 of the APC gene represents an ideal model for studying FAP
modifiers. Though carrying the same mutation, the affected subjects (45)
present with variable colonic and extracolonic manifestations which are in
several branches transmitted through the generations. Performed simulation
studies revealed a high potential of this pedigree to detect a modifier locus.
Here, the results of linkage analysis of 20 candidate regions and eventually
results on the genome wide screening for a modifier gene in FAP condition will
be presented.
P0068
Monozygotic twins showing variable expression of Muir-Torre syndrome due to
MSH2 mutation.
S. R. Forrester 1, C. Baker 2, V. Kimonis 3,
M. Schneider 1;
1Southern Illinois University School of Medicine, Department of
Pediatrics, Division of Genetics and Metabolism, Springfield, IL, 2Southern
Illinois University School of Medicine, Department of Internal Medicine,
Division of Dermatology, Springfield, IL, 33 Harvard Medical School,
Division of Genetics and Metabolism, Boston, MA.
Muir-Torre syndrome (MTS) is an autosomal dominant genodermatosis
characterized by skin tumors associated with visceral malignancies. MTS shares
many clinical similarities with Hereditary Nonpolyposis Colorectal Cancer
(HNPCC), and germ-line mutations in DNA mismatch repair (MMR) genes have also
been found in MTS families. We present monozygotic sisters with MTS caused by
a point mutation (IVS5+3A>T) in the 3’ splice site of exon 5 of MSH2
resulting in the deletion of this exon from the mRNA, thus encoding a
truncated protein. One sister developed her first squamos cell carcinoma at 50
years, and at age 53 had two sebaceous carcinomas and one adenoma removed. The
second sister had uterine cancer at age 40, metastatic colon cancer at 43,
thyroid cancer at 45, and sebaceous adenoma at 49 years. Their mother is
deceased at the age of 41 from uterine and liver cancer, and the maternal
grandfather was diagnosed with colon cancer at 50 years. Kindreds with this
same MMR mutation are described in the literature, and the types of cancers
represented in these families were quite varied and included colon, uterine,
rectal, endometrial, brain, ovarian, ureter, gastric, breast, duodenal,
sebaceous, bone, thyroid, and lung cancer. Therefore, genetic counseling must
emphasize the inability to establish any correlation between the site of the
individual mutation and spectrum of tumor types and the use of appropriate
surveillance methods. This family demonstrates the intrafamilial variability
of carcinogenesis even among monozygotic twins, and suggests that non-genetic
factors are important in modifying the expression of this syndrome.
P0069
Mutations of N- and K-Ras, p53 and FMS genes in myelodysplastic syndromes in
children
B. Jekic 1, V. Bunjevacki 1, M. Kuzmanovic 2,
I. Novakovic 1, L. Lukovic 1;
1Faculty of Medicine, Belgrade, YUGOSLAVIA, 2Institute for
Mother and Child Health, Belgrade, YUGOSLAVIA.
Myelodysplastic syndromes arise from molecular-genetic disorder of myeloid
stem cell, characterized by dysfunction of myeloid, monocytic, erythroid and
megakaryocytic lineages and high risk of evolution to ALL. Hence, MDS are
considered to be preleukemic states and are a model for studying mechanisms of
leukemic transformation. Ras, FMS and p53 were found to be the most frequently
mutated genes in adults with MDS. We have used PCR-SSCP method and sequencing
to examine mutations in these genes in 35 archival bone marrow samples of
children with MDS collected in last ten years in Institute for Mother and
Child Health. 22 DNA samples were successfully amplified with primers for
N-Ras (exons 1 and 2), K-Ras (exons 1 and 2) and FMS (region including codon
969) genes and 11 with primers for p53 gene (exons 5, 6, 7, 8 and 9). One of
analyzed samples harbored mutation in first exon, and two in second exon of
N-Ras gene. In two samples were detected mutations in second exon of K-Ras
gene. We have not detected mutations in analyzed regions of neither p53 nor
FMS genes. These findings may suggest that mutations of Ras genes play
important role in the development of MDS in children.
P0070
The relationship of chromosomes 7,9,10,17 aneuploidies and p53 gene
alterations between the low and high grade astrocytomas, using interphase FISH
technique
U. Egeli 1, T. Yakut 1, A. Bekar 2, M.
Doygun 2, E. Ogul 3;
1Department of Medical Biology and Genetics, Faculty of Medicine,
University of Uludag, Bursa, TURKEY, 2Department of Neurosurgery,
Faculty of Medicine, University of Uludag, Bursa, TURKEY, 3Department
of Neurology, Faculty of Medicine, University of Uludag, Bursa, TURKEY.
In the present study, we examined chromosomes 7, 9, 10 and 17 aneuploidies and
p53 gene alteration on surgical fresh tissue samples of 29 different grades of
astrocytomas by using flourescence in situ hybridization (FISH). Eleven of
these astrocytomas were low grade (5 pilocytic and 6 grade II) and eighteen
astrocytomas were high grade (6 anaplastic and 12 gioblastoma multiforme). All
samples were classified according to the WHO classification of tumours of the
central nervous system and none of the patients received preoperative
chemotherapy or radiotherapy. The results showed that 2 of 11 low-grade and in
6 of 18 high-grade tumours had trisomy 7. One of 11 low-grade and one of 18
high-grades had monosomy 9. Three of 11 low-grade and in 5 of 18 high-grade
tumours had monosomy 10. One of 11 low-grade and 2 of 18 high-grade tumours
had monosomy 17. Three of 11 low-grade and 6 of 18 high-grade astrocytomas had
deletion of p53 gene, although there were not monosomy 17. Based on these
findings, chromosomes 9 and 17 aneuploidies are not exclusive to low and high
grade astrocytomas. However we identified monosomy 10 and p53 deletions
similary rates between low and high grade astrocytomas, gain of chromosome 7
was identified in high-grade astrocytomas nearly two times more than low grade
ones. Thus, we suggest that loss of chromosome 10 and p53 gene abnormalities
to be earlier event than gain of chromosome 7 for carcinogenesis of
astrocytomas.
P0071
p53 mutations and PAX5 and SHB genes expression in superficial bladder
cancer
J. Mares 1, M. Babjuk 2, M. Trkova 1, J.
Duskova 3, V. Soukup 2, P. Goetz 1, Z. Sedlacek 1;
1Institute of Biology and Clinical Genetics, 2nd Medical School,
Charles University, Prague, CZECH REPUBLIC, 2Urology Clinic, 1st
Medical School, Charles University, Prague, CZECH REPUBLIC, 3Institute
of Pathological Anatomy, 1st Medical School, Charles University, Prague, CZECH
REPUBLIC.
Transitional cell carcinoma belongs to a very heterogenous group of neoplasms.
Prognosis of a patient at the time of diagnosis is a basic problem of the
bladder cancer therapy and has encouraged the search for prognostic markers.
The role and possible oncogenic activity of the PAX genes have been discussed
recently. A candidate gene PAX5 is situated on the 9p21-23, the region of the
most frequent genetic changes in bladder cancer, as well as another signal
transduction gene SHB. Concerning the prognostic potential of p53 mutations,
recent research yielded contradictory results. The aim of the study was to
define new combinations of prognostic markers reflecting biological behaviour
of individual tumours in order to identify patients at risk for tumour
progression. We investigated 44 patients with superficial bladder cancer and
20 controls for p53 mutations in exons 5-9 and adjacent intronic sequences by
the SSCP and direct genomic sequencing. One mutation (del 128 Pro) was
detected among the 44 tumours (2.3 %). 36 patients overexpressed at least one
of the PAX5 or SHB genes, 26 of them overexpressed both genes. The staging and
grading of these pacients were generally higher then of those without
increased expression. The correlation of PAX5 and SHB expression and clinical
and histopathological data was evaluated. In conclusion, our results indicate
that combination of expression data might be used as a diagnostic tool in
superficial bladder cancer. Supported by the grant IGA MZ NC/5961-3.
P0072
Loss of heterozygosity of p53 gene in gastic carcinoma in the region of
easten Turkey
I. Pirim 1, A. Karaman 2, M. Ikbal 1;
1Ataturk Universty, Erzurum, TURKEY, 2State Hospital,
Erzurum, TURKEY.
Loss of heterozygosity effecting various chromosomes has been characterized on
tumor of many human cancers. Tumor suppressor gene p53 was found to be primer
target for that losses. In our study, we examined 41 patients with gastric
neoplasm for loss of heterozygozity effecting the p53 gene by using PCR/RFLP
technique. The samples were run on to agarose gel and visualized on UV.
Cancerous lesion of wet tissues from 25 of 41 patients was taken together with
their peripheral blood samples. 6 patients was inoperable, so only blood was
taken and paraffin tissue of 10 patients were examined for allelic losses. The
PCR was carried out by using two sets of primers, both amplified 72. codon of
exon 4 of p53 gene. The primer called G-H gave amplified fragment of 66 bp and
the other I-J gave 247bp fragment. These fragment were subjected to
restriction enzyme BstUI for detecting LOH. 18 out of 41 patients exhibited
heterozygosity loss 43,6 % and many of these LOH positive cases had lenf
metastasis. We could not determined any relation between p53 LOH positivity
and sex or age. Finally, it has been shown that LOH in p53 gene are common in
gastric cancer and play important role for cancer progression.
P0073
Loss of heterozygosity in tumours of carriers of germline TP53
mutations
P. Goetz 1, M. Trkova 1, L. Foretova 2,
V. Krutilkova 3, R. Kodet 1, J. Mares 1, Z.
Sedlacek 1;
1Charles University, Prague, CZECH REPUBLIC, 2Masaryk
Memorial Cancer Institute, Brno, CZECH REPUBLIC, 3University Hospital
Motol, Prague, CZECH REPUBLIC.
A strong genetic determination is observed in about 5% of all cancer cases.
These patients belong to families with high cancer incidence and/or suffer
from early onset tumours or multiple or multifocal malignancies. Many cases of
hereditary predisposition to cancer are due to a germline mutation in one of
the tumour suppressor genes. Some familial cancer syndromes show
predisposition to a particular type of cancer (e.g. breast or colon cancer).
The much rarer Li-Fraumeni syndrome (LFS) is distinct because members of LFS
families suffer from a wide spectrum of different malignancies including
sarcomas, brain tumours, breast cancer, adrenocortical carcinomas and other
tumours. The cancer predisposition in most of these families is due to a
germline mutation in the TP53 gene. It is generally expected that the tumours
in most of such predisposed persons arise after the wild type TP53 allele is
lost in a particular cell clone in a carrier individual. We show on our
material that many tumours from germline TP53 mutation carriers retain the
wild type TP53 allele. The development of these tumours in LFS individuals
must therefore be based on another mechanism of the TP53 gene silencing than
simple DNA loss. Alternatively, one functional TP53 allele may still be
present in these tumours. We also compare these observations with records in
our web database of published germline TP53 mutations, which is a very useful
tool for different analyses of this intriguing syndrome. Supported by grant
IGA MZ CR NC/6513-3.
P0074
Gene expression following tet-regulated reexpression of wt p53 in lung cancer
cells
I. Wieland 1, A. Brüning 2, H. Burtscher 3,
U. H. Weidle 3;
1Otto-von-Guericke University, Magdeburg, GERMANY, 2Institute
for Cell Biology (Cancer Research), University Essen Medical School, Essen,
GERMANY, 3Roche Diagnostics GmbH, Penzberg, GERMANY.
The tumor suppressor p53 is inactivated in a wide range of human tumors. In
non-small cell lung carcinoma (NSCLC) cell line NCI-H358 p53 and
p16INK4a/p15INK4b are deficient while Rb is expressed. This condition occurs
frequently in native human NSCLC and, therefore, appears to be particularly
suited for studying the effects of reexpression of wild-type (wt) p53 in lung
cancer cells. We generated the wt p53 inducible NSCLC line H358B22 using a
tetracycline/doxycline-regulated (tet-on) expression system. High-level
reexpression of wt p53 suppressed proliferation of H358B22 cells completely.
Most growth arrested cells stayed viable over a period of 1 week. Therefore,
p53 appears to function mainly as an inducer of cell cycle arrest rather than
as an inducer of apoptosis in these cells. This growth inhibitory effect of wt
p53 is reversible after 24 h of p53 induction, but it becomes irreversible
after 48 h of wt p53 induction followed by resilencing of the exogenous wt
p53. Therefore, genes regulated in growth arrested H358B22 cells were
investigated by microarrays (Affymetrix), RT-PCR and Western blotting. Most
differences in gene expression were reversible upon resilencing of exogenous
wt p53. However, in irreversibly arrested H358B22 cells a subset of genes
including Bax, Fas, p27KIP1, p21WAF1, B-myb, cyclin A and IGF-BP3 escaped
reversibility.
P0075
GSTM1 null, GSTT1 null, GSTP1 (Ile105Val) and CYPA1 (T6235C) Genotypes in
Childhood Acute Leukemia
G. Balta 1, E. Ozyurek 1, U. Ertem 2, G.
Hicsonmez 1, C. Altay 1, A. Gurgey 1;
1Hacettepe University, Pediatric Hematology Unit, Ankara, TURKEY, 2Sami
Ulus Children's Hospital, Ankara, TURKEY.
The purpose of the present study is to elucidate the role of GSTM1 null, GSTT1
null, GSTP1 (Ile105Val) and CYPA1 (T6235C) polymorphisms in the etiology of
childhood acute leukemia. The study showed that: A) Frequencies of the
genotypes were almost identical in 145 ALL patients and 186 healthy controls.
Differences in the frequencies were not statistically significant in all
genotypes (>p 0.2). The frequency of GSTM1 and GSTT1 double null genotype
was lower in ALL (9.9%) than controls (13%). In ALL patients: 1- No
statistically significant differences were found in the frequencies of
genotypes between patients belonging to B cell (73) and non B cell lineage
(41), yet the frequency of GSTT1 genotype was lower in the group non-B cell
(17%) than B cell and control (23%). 2- No differences were found in
frequencies of the genotypes between male and female patients. 3- There was no
differences in distribution of the genotypes among age groups, except
frequency of the GSTT1 genotype was lower in patients 10-17 years (17%) than
0-2, 2-9 years (23%) and control. 4- The frequency of CYPA1 polymorphism was
statistically significant in group of patients with WBC count 10.000-50.000 at
presentation (58%) than <10.000 (20%), >50.000 (21%) (p 0.01) and
control (29%). Frequency of GSTT1 genotype was lower in patients with
>50.000 (10%) than others and control (23%). B) Statistically significant
association was found in the frequencies of GSTT1 genotype between AML
patients (3.4%) and control (23%) (p 0.016) while no association was observed
for other genotypes.
P0076
Increased accuracy of leukemia diagnosis by combined analysis of morphology
and FISH using the Duet automatic cell scanning system.
C. Kaplinski 1, I. Hardan 1, M. Daniely 2,
A. Toren 1, A. Shimoni 1, A. Avigdor 1, M.
Reichart 2, A. Nagler 1, T. Kaplan 2, F.
Brok-Simoni 1, G. Rechavi 1, N. Amariglio 1, L.
Trakhtenbrot 1;
1Dept. of Pediatric Hemato-Oncology and Inst. of Hematology, The
Chaim Sheba Medical Center, Tel-Hashomer, ISRAEL, 2BioView Ltd.,
Rehovot, ISRAEL.
Fluorescence in situ hybridization (FISH) is a valuable tool in clinical
practice of leukemia. However, high false positive and false negative rates
complicate the interpretation of results. These limitations are especially
important in follow-up examinations, and in detection of minimal residual
disease (MRD). Recently, the Duet scanning system (BioView Ltd, Rehovot,
Israel) was introduced. The system provides two important features: Automatic
scanning of large number of cells, and combined analysis of morphology and
FISH on the same cells. Prior to scanning, blood samples are processed
according to a unique protocol, which allows removal of RBC and 2 consecutive
staining of WBC (giemsa or immunocytochemistry and FISH). This approach was
applied to 80 samples of various hematological malignancies in order to: a)
Determine the lineage of cells carrying specific chromosomal rearrangement. b)
Enhance FISH analysis accuracy in leukemic cells. c) Determine clonality and
maturity of residual recipient/donor cells in bone marrow transplantation. d)
Determine the maturity of cells carrying chromosomal rearrangements in MRD.
The results were compared to the diagnosis given by routine methods. We found
that the Duet system enabled increased specificity and sensitivity of leukemic
cells detection. Scanning automatically large numbers of cells provided rapid
and efficient identification of rare cells in MRD cases (up to one leukemic
cells in 15,000 WBC). The combined morphologic and FISH information of
suspected cells enhanced the specificity of leukemic cells detection and
reduced FP drawbacks. These preliminary results indicate the advantage of
using such approach in diagnosis of leukemic diseases.
P0077
Are Fanconi Anaemia Genes Inactivated in Sporadic Acute Myeloid
Leukemia?
M. D. Tischkowitz 1, N. V. Morgan 1, C. Eddy 1,
S. Ball 2, S. E. Langabeer 3, I. Vorechovsky 4, R.
Stoeger 1, D. Grimwade 1 ,3, C. G. Mathew 1,
S. V. Hodgson 1;
1GKT School of Medicine, London, UNITED KINGDOM, 2Department
of Haematology, St George's Hospital, London, UNITED KINGDOM, 3Department
of Haematology, University College, London, UNITED KINGDOM, 4Department
of Bioscience, Karolinska Institute, Stockholm, SWEDEN.
Fanconi Anaemia (FA) is an autosomal recessive disorder characterised by
congenital abnormalities, defective haemopoesis and a greatly increased risk
of Acute Myeloid Leukaemia (AML). We are investigating whether mutations in
the FA genes might predispose to the development of sporadic AML. Quantitative
fluorescent PCR was used to screen archival DNA from peripheral blood or bone
marrow from AML cases for deletions in the cloned FA genes,
FANCA, C, D2,
E, F, G. Of the 103 samples successfully screened for the
FANCA
gene, four heterozygous deletions were found (see table). Sequence analysis of
the other allele in these four cases did not locate a second mutation. A
sodium bisulphite conversion assay was developed to detect methylation of the
FANCA
CpG island. There was no evidence of allele inactivation by hypermethylation
in these 4 samples, nor in a further 28 non-deleted samples. No deletions were
found on screening the
FANCC,
D2,
E,
F and
G
genes in 64, 68, 31, 42 and 51 samples respectively.
FANCA is a large
gene (43 exons, 80kb) with a high incidence of deletion mutations in affected
FA patients. These results show that such deletions may also be found in
sporadic AML and may have contributed to leukaemogenesis.
Characteristics of FANCA deletion samples (*=
deletions endpoints undefined)
| Sample |
Type |
Cytogenetics |
FANCA
Deletion |
| 1 |
Male 65
yrs
FAB M2 |
43,XY,del(5)(q15q3?3),-6,-7,
r(7),i(8)q),add(16)(q?24),-17,-22,
+der(?)t(?;6)(?;?p11) |
heterozygous
ex5-43* |
| 2 |
Female 45
yrs
FAB M1 |
44.XX,add(2)(q2),add(5)(q?),-7,
+?10, -12, add(16)(q2), -18, -20, +mar[4] |
heterozygous
ex19-21 |
| 3 |
Male 56
yrs
FAB M6 |
44,XY,del(1)(q21q25),add(4)(q?
25),-5,-7,-11,-12,del(12)(q21q24),add(13)(q13), add(16) |
heterozygous
11-21 |
| 4 |
Female 69
yrs |
N/A |
heterozygous
ex 5-43* |
P0078
Gene expression patterns in childhood acute lymphoblastic leukemia
H. Bruchova, R. Brdicka;
Institute of Hematology and Blood Transfusion, Department of Molecular Genetics,
Prague 2, CZECH REPUBLIC.
Array hybridization technique represents a useful method for the expression
profiling of large gene sets during disease processes. Using this technology
we studied gene expression in childhood acute lymphoblastic leukemia (ALL)
patients. For detection of transcription activity we used Human Cancer cDNA
Nylon Arrays (Clontech, USA) with 588 genes that can be involved in
transformation. Total RNA was isolated from bone marrow leukocytes of patients
at the time of diagnosis (previously untreated). The standard sample was
prepared by RNA mixing of control individuals (bone marrow donors). Our
objectives were to identify genes that were differentially expressed in ALL
and might contribute to the disease development (and characterization).
Obtained gene expression profiles of patients were compared with the standard
profile. The majority of patient genes showed the similar gene activity as in
the control sample (e.g. gluthatione-S-transferase homolog, vimentin, rho-GAP
hematopoietic protein C1, rho GDP dissociation inhibitor 2, fau etc.). Many
genes displayed significant expression changes only in some patients. In a few
genes it was possible to observe similar significant changes of gene
expression in most patients ( eg. PCNA, MMP8). These changes might be
associated with common stream of the disease process and they can be studied
in more detail. Supported by the grant IGA MZ CR no. NM/5901-3.
P0079
The relationship between the chromosomal rearrangement complexity and disease
agresivity in some cases of leukemia
A. G. Lungeanu 1, A. Arghir 2, A. Lupu 3,
D. Mut-Popescu 4, N. Berbec 4, L. Popescu 5;
1National Institute, Bucharest, ROMANIA, 2"Victor
Babes" Institute, Bucharest, ROMANIA, 3"Carol
Davila"University, Bucharest, ROMANIA, 4"Carol Davila"
University, Bucharest, ROMANIA, 5"Carol Davila" University,
"Victor Babes" Institute, Bucharest, ROMANIA.
Among over 100 patients with myeloid and lymphoid leukemias investigated
cytogeneticaly during the last 15 months, in four cases, disease evolution was
determined by the complexity and nature of chromosomal abnormalities
identified at the first presentation. First case, a 22 years old man with
L3type ALL, exhibited: del 3q26;del 5p13; t(8;14)(q24;q13);del 9p11q11 and inv
15p12qter in all cells from bone marrow. He died after four months. The second
case, a woman of 62 years old with acute leukemia weak-differentiated,
refractory to treatment, showed 48-54 chromosomes and 3-4 markers derived from
chromosomes 5 and 12. She died in the next three weekes. The third case, a
young man of 27 years old, with acute myeloid leukemia, apart of Ph chromosome
presented del11q21 and del16q22. The rapid death of the three cases was a
powerful prove of positive correlation between the complexity of chromosomal
changes and disease agresivity. In change, a constitutional translocation
t(3;5)(q26;q21) identified in a 72 years old woman with ET, conferred
favourable evolution of the disease after a succesfull treatment with HU. So,
we appreciate that, if in the first three cases of myeloid and lymphoid
leukemias could be a direct relationship between the complexity of genomic
rearrangements identified at the onset and agresive development of the
disease, in the fourth case of ET, constitutional translocation t(3;5), seems
to be not involved in the etiology of the disease.
Acknowledgements: VIASAN project 089, ANSTI 5195/1999-2001, and
Schering-Plough Central East Ag.
P0080
Expression of Negative Regulators of Cell Cycle in Human Acute Leukemia
Cells.
D. Szczesniak 1, J. Kocki 1, M. Cioch 2,
A. Dmoszynska 2, B. Marzec 1, J. Wojcierowski 1;
1Department of Medical Genetics, Medical Academy, Lublin, POLAND, 2Department
of Hematology, Medical Academy, Lublin, POLAND.
The negative regulators of cell cycle like cyclin dependent kinases inhibitors
genes, Rb family genes and p53 gene play important role as inhibitors of cell
proliferation. Incorrect expression of these genes may cause disturbances in
cell machinery, uncontrolled cell division and consequently malignant
transformation.In our research we examined the level of cell cycle negative
regulators genes expression on mRNA level in bone marrow samples in patients
with acute leukemia before treatment.For detection of mRNA we used the Multi
Probe RNase protection Assay System (RiboQuant). We analyzed the expression of
cyclin dependent kinases genes (p16 and p21 family), Rb family genes and p53
gene.Obtained results show significantly high level of p53, p27, p19 and p18
mRNA, while the level of Rb and p16 mRNA is very low in the examined cells.The
correlation of the results with the level of other cell cycle regulators
expressions and clinical data may give us important information about new
prognostic factors in hematological malignancies.
P0081
Submicroscopic deletion at the breakpoint in chromosome der(9) in Ph+ acute
lymphoblastic leukemia (ALL)
I. F. Loncarevic-Barcena 1, B. Shell 1, H. J.
Fricke 2, M. Prechtel 1, M. Ziegler 1, U. Claussen 1;
1Humangenetik und Anthropologie, Jena, GERMANY, 2Klinik
für Innere Medizin II, Jena, GERMANY.
Submicroscopic deletions in the breakpoint region of chromosome der(9)t(9;22)
are found in ~25% of patients with chronic myeloid leukemia (CML). Notably,
these deletions are strongly associated with a shorter life expectancy when
non transplanted CML patients are compared. We present molecular and clinical
data of a 22 year old male patient with a t(9;22) positive B-cell specific
acute lymphoblastic leukemia (B-ALL) that exhibit a deletion in chromosome
der(9). In ALL this deletion was first and uniquely detected so far in one of
48 ALL patients investigated by Reid et al (abstract: 1334, ASH meeting 2001).
The rare occurrence of this deletion in ALL makes it difficult to evaluate the
clinical impact. The deletion we found is located proximal to the breakpoint
in der(9)t(9;22). RT-PCR detected a b3a2 BCR-ABL and failed to detect an
ABL-BCR transcript. No response to therapy was achieved with a high dose
protocol (Hölzer Studie 5/93). The patient was subjected to salvage therapy
with Idarubicin/AraC and reached a partial remission with 20-25% BCR-ABL
positive cells at day 125 after initial therapy. At present, STI571 is
administered and a stem cell donor is searched. The data confirm that
deletions in der(9) can also be found in Ph+ ALL. The clinical significance of
this rare deletion in ALL is unknown and has to be evaluated by increasing the
study cohort.
P0082
Acute monocytic leukemia and multiple abnormalities in a child with duplication
of 1q detected by GTG-banding and SKY.
M. R. Baruffi 1, C. A. Scrideli 2, J. A. Squire 3,
J. Karaskowa 3, E. S. Ramos 4, B. Heck 4, L. G.
Tone 4;
1Ribeirao Preto Medice School, University of Sao Paulo, Ribeirao
Preto, SP, BRAZIL, 2Ribeirao Preto Medicine School, University of Sao
Paulo, Ribeirăo Preto, SP, BRAZIL, 3University of Toronto, Toronto,
ON, CANADA, 4Ribeirao Preto Medicine School, University of Sao Paulo,
Ribeirao Preto, SP, BRAZIL.
Patients with 1q duplication have demonstrated a wide range of multiple
congenital abnormalities, but a clinical delineation of a trisomy 1q “syndrome”
was proposed. Alterations involving this same chromosomal region have also
being described in various hematopoietic malignant disorders and a series of
candidate genes that may be associated with neoplasia have been described in
this region. We describe a female girl with low birth weight, microcephaly,
mid facial hipoplasia, synophris, short palpebral fissures, epicanthic fold,
beak-like nose, narrow palate, teeth abnormalities, cardiac defect,
syndactily, and motor delay, that presented, at 18 months of age, an acute
monocytic leukemia (FAB-M5) according to cytological, histochemical and
immunophenotyping features. The patient failed to achieve remission, and died
2 months after diagnosis. Cytogenetic study of the bone marrow cells by
GTG-banding showed:
44~48,XX,-X[9],dup(1)(q23q44)[35],+2[27],-6[13],+7[4],-8[3],-9[7],+9[2],+10[3],+11[13],
+12[5],-14[5],-15[4],-17[7],-18[13],-19[3],+21[5],-22[5],+22[2],+mar[5]cp[35].Spectral
karyotyping (SKY) was also performed to identify the aberrations
46,XX,der(1)dup(1)(q23q44)t(1;1)(p36;q32),der(6)t(6;8)(p25;q13),+11,der(11)t(11;18)(q10;q10).Peripheral
blood cytogenetic analysis was not performed due to repeated blood products
transfusions and the precocious patient death. The dismorphological features
with the dup(1q) founded in all bone marrow cells analyzed suggest that this
is probably a constitutional chromosome alteration and the first, in our
knowledge, association of a trisomy 1q"syndrome" with AML.
Supported by: FAEPA, FAPESP, CCS, NCIC
P0083
Three new cases of complex Ph' variants in Chronic Myeloid Leukemia
A. Carrió 1, D. Costa 1, A. Arias 1, R.
Queralt 1, F. Cervantes 2, J. Aguilar 3, D.
Colomer 3, E. Campo 3;
1Servei de Genčtica. CDB. Hospital Clínic, Barcelona, SPAIN, 2Servei
d'Hematologia. Hospital Clínic, Barcelona, SPAIN, 3Unitat
d'Hematopatologia. CDB. Hospital Clínic, Barcelona, SPAIN.
A 90-95% of patients diagnosed with Chronic Myeloid Leukemia (CML) show the
Philadelphia chromosome (Ph) as a result of the t(9;22)(q34;q11). About 5-10%
of CML show the variant forms: simple (22q11qter is translocated into a
chromosome other than 9) and complex (three or more chromosomes are involved).
We present cytogenetic, fluorescence in situ hybridization (FISH), and
molecular analyses in three cases of the complex variant.
The chromosome bands involved were 11q13 (two cases) and 1p36.1. The FISH
analyses (locus specific, centromeric and whole chromosome painting) showed
that the bcr/abl fusion gene was in chromosome 22 in all three cases,
suggesting a complex variant rather than a clonal evolution.
P0084
Characterisation of Acute Myeloid Leukemias (AML) with complex aberrant
karyotype using gene expression analysis and mutation screening of candidate
genes
S. Mergenthaler, C. Schoch, S. Schnittger, A. Kohlmann, W. Kern, M.
Dugas, M. Klaus, J. Christodoulou, W. Hiddemann, T. Haferlach;
Labor für Leukämiediagnostik, Klinikum Grosshadern, Munich, GERMANY.
AML represent a pathogenetically and prognostically heterogeneous group. For
differentiation of AML-subgroups, cytogenetics offers the most evaluated and
established criteria today. 10-15% of AML-patients show complex aberrant
karyotypes in leukemic blasts, associated with a most adverse progression of
the disease. So far, no crucial candidate genes relevant for the pathogenesis
were identified.
Data obtained by 24color-FISH and CGH in 50 AML-cases with complex aberrant
karyotype demonstrated a much larger percentage of loss than gain of genetic
material. Frequent observations included deletions of the entire chromosomes 5
and 7, as well as interstitial deletions within their long arms.
These deletions may represent the first of two required mutation events to
deplete a tumorsuppressor gene’s function. Alternatively, haploinsufficiency
with only one mutation event might already be sufficient to reduce the
physiologically necessary amount of gene product.
To evaluate both models, we currently investigate 25 of these AML-cases with
complex aberrant karyotype more precisely on molecular genetic level:
utilizing gene expression analysis (GeneChip U133) and mutation screening
(Single Strand Conformation Polymorphism Analysis, SSCPA) we focus on 20
functionally relevant candidate genes on chromosomes 5 and 7, involved in
apoptosis, cell cycle/transcription regulation and DNA repair mechanisms.
So far, SSCPA in 25 AML-patients and 10 healthy controls using different
conditions excluded the General Transciption Factor GTF2H2 (5q12.2-q13.3),
involved in transcription/transcription-mediated DNA-repair, as a major
candidate gene in complex AML-pathogenesis.
Our future investigations aim at providing a deeper insight into basic
pathogenetic mechanisms of complex AML with subsequent implementation in
prognosis and therapy strategy.
P0085
Validation of human BAC clone microarray based CGH studies in HL-60 cell
line.
M. Alkan 1, C. Ulger 1, G. A. Toruner 1,
M. Muhammed 2, S. Damani 2, P. Tolias 1 ,3,
M. Schwalb 1, J. Dermody 1;
1Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey
Medical School, Newark, NJ, 2Spectral Genomics, Houston, TX, 3Center
for Applied Genomics, Public Health Research Institute, Newark, NJ.
Comparative genomic hybridization (CGH) is a method, used to detect, and map
DNA sequence copy number differences between two genomes in a single
experiment. Although it is a valuable technique, the lower resolution of CGH
compared to molecular genetic techniques have limited its application. The
utilization of microarray technology in CGH analysis has the potential to meet
these challenges. For this purpose, a well-characterized promyelocytic cell
line HL-60 was analyzed by using Human BAC Array- 3MB system, which was
developed by Spectral Genomics TM. The glass array is composed of
1003 non-overlapping BAC library clones which encompass the human genome with
a resolution of 3 Megabases. In HL-60, amplification of the region of 8q24, an
extra copy of chromosome 18, and deletions in loci of 5q11.2-q31, 6q12, 8p23,
9p21.3-p22, 10p12-15, 14q22-q31, 16q21, and 17p12-17p13.3 were detected. These
results are largely concordant with the previously reported aberrations, and
indicate that this system might be an alternative to conventional CGH
analysis.
P0086
High-throughput tissue microarray analysis of 11q13 genes amplification
(CCND1, FGF3/FGF4, FGF3, EMS1) in urinary bladder cancer
B. Zaharieva 1, R. Simon 2, T. Gasser 3,
G. Sauter 2, D. Toncheva 1;
1Department of Medical Genetics, Medical Faculty Sofia, Sofia,
BULGARIA, 2Institute of Pathology, University of Basel, Basel,
SWITZERLAND, 3Urologic Clinics, Cantonal Hospital Liestal, Liestal,
SWITZERLAND.
Gene amplification is a common mechanism for oncogene overexpression.
High-level amplifications at 11q13 had been repeatedly found in bladder cancer
by comparative genomic hybridization and by other techniques. Putitative
candidate oncogenes located in this region are CCND1, EMS1, FGF3 and FGF4. To
evaluate the involvement of these genes in bladder cancer, we screened a
tissue microarray (TMA) containing 2317 samples by FISH. Among all tumors with
11q13 amplifications (13.3%) 68.3% had all 4 genes amplified, 19.5% had
amplified CCND1, FGF4 and FGF3 together, 0.8% had FGF4, FGF3 and EMS1
coamplified. Single amplification of CCND1 was found in 9% of the tumors,
while 1,6% had single amplification of EMS1 and 0.8% - of FGF4 suggesting that
CCND1 is the major target gene in the 11q13 amplicon in bladder cancer. The
frequency of both gains and amplifications of all genes increased
significantly from stage pTa to pT1-4 and from low to high grade tumors.
Increased copy number changes of all 4 genes were associated with survival of
patients with tumors from all stages and progression of pT1 tumors and were
not associated with recurrence of pTa tumors and survival of patients with
pT2-4 tumors. Tumors with gains of FGF3, FGF3/FGF4 and EMS1 were shown to
behave like tumors with amplifications rather than like normal tumors contrary
to CCND1 where gained tumors behaved like CCND1 normal rather than like CCND1
amplified tumors.
P0087
Dystrophic Scoliosis And Genetic Polymorphisms In Patients With
Neurofibromatosis
S. Funke 1, E. Morava 2, M. Czakó 3, B.
Cser 4, G. Kosztolányi 4, T. Illés 5;
1Dep. Medical Genetics and Dep. of Obstetrics and Gynecology,
University of Pécs, Pécs, HUNGARY, 2University of Pécs, Pécs,
HUNGARY, 3MTA-PTE Clinical Genetic Research Group, University of
Pécs, Pécs, HUNGARY, 4Department of Medical Genetics and Child
Development, University of Pécs, Pécs, HUNGARY, 5Department of
Orthopedics, University of Pécs, Pécs, HUNGARY.
The dystrophic form of scoliosis in neurofibromatosis 1 (NF1) is often
associated with severe decrease in bone mineral density, significantly
hindering reconstructive bone surgery. Osteoporosis has been found to be
associated with distinct polymorphisms of the vitamin D receptor gene (VDR),
oestrogen receptor gene (OER) and the collagen 1a1
gene (COL1A1) in the general population. The purpose of the present
case-control study was to evaluate the hypothesis, whether the genotypes at
these three polymorphic loci are associated with decreased bone mineral
density in scoliotic patients with neurofibromatosis. Genotype of 21 selected
NF1 patients with scoliosis and decreased bone mineral density was compared to
21 patients with non-scoliotic NF1 with normal bone density. Patients with
idiopathic scoliosis with normal bone density measurements (21) have been also
assessed for the same genetic polymorphisms. In this pilot study of altogether
63 individuals no association was found between VDR and OER polymorphisms, and
the phenotype. The genetic marker distribution in the idiopathic scoliosis
(IS) group did not significantly differ from that of scoliotic NF1 patients.
However, we observed a threefold prevalence of the homozygous polymorphism
(CC) over the heterozygous form (Cc) of the COL1A1 gene in non-scoliotic NF1
patients compared to patients with scoliosis presenting with an almost equal
distribution in this genotype. This difference was not statistically
significant. The sample size of this pilot study is not large enough to draw a
final conclusion. A possible protective role of CC genotype in non-scoliotic
NF patients deserves reevaluation in a larger group of patients.
P0088
Radiological appearance of intracranial tumours in neurofibromatosis
(NF1)
E. Leisti;
Oulu University Hospital, Department of Radiology, Oulu, FINLAND.
In a population-based study on neuroradiological imaging of individuals with
NF1, 10 of 124 studied patients (8%) presented with intracranial tumours other
than optic gliomas or T2 hyperintense lesions. Six patients aged 6 to 53 years
had an astrocytoma, one patient had a suspected astrocytoma which proved
histologically to be normal brain tissue, one patient had a small lipoma in
the interpeduncular cistern, one patient had a hypophyseal adenoma and one
patient presented with an enhancing lesion of the cavernous sinus. - The
astrocytomas showed wide variation in their behaviour and MR imaging. Four of
the tumours were progressive, one histologically confirmed astrocytoma
disappeared spontaneously and one astrocytoma appeared within a previously
detected T2 hyperintense lesion, which was partly located in the region of the
optic radiation and had remained stable for several years. The results
indicate that the fate of an astrocytoma in NF1 cannot be predicted on the
basis of one imaging only, but that the patients need close follow-up.
P0089
Do some additional chromosome rearrangements mean a favourable T-cell
prolymphocytic leukemia prognosis with preserved alkylator based treatment
sensitivity?
N. Kokalj Vokac 1, S. Zver 2, A. Erjavec 1,
B. Zagradisnik 1, A. Zagorac 1, D. Zontar 2, P.
Cernelc 2;
1Maribor Teaching Hospital, Maribor, SLOVENIA, 2Univ.Clinical
Center Ljubljana, Ljubljana, SLOVENIA.
A 77-year-old woman came to haematological department because of leucocytosis,
(WBC 276x109/L; Hgb 111 g/L, Plt 239x109/L), sweating and weight loss. Bone
marrow biopsy revealed 80% lymphoid-cell infiltration. Morphologically cells
appeared as prolymphocytes and immunohistochemically they were CD3, CD4, CD5
positive, while being CD8, CD20, CD30, CD43, CD56, TIA-1 and Granzyme B
negative. Flow-cytometrically performed T-lymphoid immunological markers CD2,
CD3, CD4, CD5 and CD7 were highly, more than 90% positive. Diagnosis of T-cell
prolymphocytic leukemia (T-PLL) was made and the later was confirmed also by
cytogenetic analysis. T-PLL specific chromosome rearrangements were observed:
inv(14)(q11q32), i(8)(q10) and del(11)(q22q23) involving ATM gene. Complex
translocation of X chromosome, probably involving MTCP1 gene, was present on
der(X)t(X;3)(q28;p25)t(X;16)(p14;q12). There were several other chromosome
rearrangements observed, including chromosomes 5, 6, 13, 14, 17, 20 and 22.
Classical cytogenetic analysis was confirmed by FISH, using Cytocell
Octochrome Multiprobe System, and some locus specific DNA probes.
T-PL leukemia is aggressive, and refractory to alkylator-based therapy, with a
median survival of 7 months. Treatment options are highly immunosupressive
2-CDA, Pentostatin or Campath 1-H. Because of the patient's advanced age, we
have started with chlorambucil 10 mg/m2 for 5 consecutive days, repeatingly
every 4 weeks. After 4 cycles of chlorambucil the patient was without any
clinical symptoms, with WBC 23,4x109/L, Hgb 128g/L, Plt 256x109/L. To our
knowledge, the additional chromosomal rearrangements: der(6)t(X;6)(p14;q25),
der(13)t(13;14)(q22;q11), t(5;13)(q34;p11), r(17)(p13q21), t(17;20)(q21;q13),
22p+ , were not yet described in the literature. Some of them may means
favourable T-PL prognosis because of preserved alkylator agent treatment
sensitivity.
P0090
Prognostic Significance Of Small Cell Clones With Hyperdiploidy In Childhood
Acute Lymphoblastic Leukemia (all).
Z. Zemanova 1, K. Michalová 1 ,2, L.
Sindelarova 1, J. Brezinova 2, S. Kurkova 2, P.
Smisek 3, O. Hrusak 4, J. Stary 3;
1Center of Oncocytogenetics, General Faculty Hospital and 1st Medical
Faculty of Charles University, Prague, CZECH REPUBLIC, 2Institute of
Haematology and Blood Transfusion, Prague, CZECH REPUBLIC, 32nd
Department of Pediatrics, Faculty Hospital Motol, Prague, CZECH REPUBLIC, 4Institute
of Immunology, 2nd Medical Faculty of Charles University, Prague, CZECH
REPUBLIC.
Children with ALL and chromosome hyperdiploidy in bone marrow cells have a
better prognosis in contrast to those with other cytogenetic abnormalities.
Therefore early detection of hyperdiploid cell clones is important and can
lead to appropriate less aggressive therapy protocol with the lower risk of
the late side effect. For the assessment of hyperdiploidy we use consecutive
double target interphase FISH (I-FISH) with combination of alpha-satellite
and/or locus-specific probes for 10 chromosomes most frequently
overrepresented in hyperdiploid clones (200 interphase nuclei analysed per
slide and probe-mix, cut-off level 2.5% tested on controls, standard deviation
not exceed 0.5%). I-FISH is quick and sensitive screening method which enables
to find even "hidden" small pathological clones. Structural and/or
complex chromosomal aberrations in hyperdiploid cells were analysed by
multi-color FISH (mFISH).
During the last four years we examined prospectively or retrospectively 88
children with ALL (56 boys, 32 girls; mean age 8 years). Various level of
hyperdiploidy was found in 60 children (68%). The extent of pathological
clones being 2.5 - 100%. Small clones under 10% were detected in 18 patients
(20,5%). Structural or complex chromosomal rearrangements together with
hyperdiploidy were found in 23 patients (26%). We compare FISH and cytogenetic
findings, results of DNA analysis by flow cytometry and clinical course of the
disease in all patients with a particular respect to the prognostic
significance of small pathological clones and complex chromosomal
rearrangements.
This work was supported by grants IGA MZ CR NE 6472-3, GACR 301/01/0200 and
IGA MZ CR 6406-3.
P0091
Cytogenetic studies in T-cell acute lymphoblastic leukemia: a report of 35
cases.
N. Douet-Guilbert 1, M. J. Le Bris 2, A. Herry 2,
F. Morel 2, G. Le Calvez 3, V. Marion 3, J. F.
Abgrall 3, C. Berthou 1, M. De Braekeleer 2;
1Service d'Hématologie clinique, CHU, Brest, FRANCE, 2Service
de Cytogénétique, Cytologie et Biologie de la Reproduction, UBO & CHU,
Brest, FRANCE, 3Service d'Hématologie biologique, CHU, Brest,
FRANCE.
A total of 198 patients with acute lymphoblastic leukemia (ALL), including 189
at diagnosis and 9 at relapse, had a cytogenetic analysis on a bone marrow
sample between 1981 and 2001. Thirty-five ALL (17.7%) were of the T cell
lineage. The immunophenotyping performed on 32 cases showed that 30 were true
T-cell ALL, one was mixed T-cell/B-cell and one mixed T-cell/Myeloid. The 35
patients were distributed in 14 children and 21 adults. The quality of the
chromosomal preparations was too poor to allow a feasible identification in 2
cases and one culture did not yield metaphases. Karyotyping was sucessfully
performed in 32 patients. A normal karyotype was observed in 5 of the 13
pediatric cases (38.5%) and in 5 of the 19 adult cases (26.3%). These values
are within the range observed in other series of T cell-ALL. Numerical
chromosome abnormalities were rare, 77.3% of the abnormal karyotypes (17/22)
being pseudodiploid. Translocations involving band 14q11 were observed in 7
patients whereas band 12p13 was deleted in 2 cases and translocated in a
further 3 cases. The short arm of chromosome 11 was involved in 4
translocations, band 11p13 in 2 and band 11p15 in another 2 cases [t(4;11) and
t(1;4;11)]. Other recurring structural rearrangements include del(6q) in 3
cases and del(5q) in 2. Most of these recurrent abnormalities are different
from those of B-lineage ALL. Some are known to involve T cell receptor genes
whereas others can lead to the discovery of new genes that are important to
T-lineage leukemogenesis.
P0092
Detection of Philadelphia Chromosome in Chronic Myelogenous and Acute
Lymphoblastic Leukemia in two locations in Ecuador.
J. C. Ruiz-Cabezas;
Hospital Dr. Juan Tanca Marengo SOLCA, Guayaquil, ECUADOR.
A previous study sustained that there may be a difference in the presence in
Philadephia (Ph) Chromosome t(9;22) in the studied ecuadorian series due to
the ethnical content and geographical location (Quito, 2800m) of the studied
human group.
We present here data that supports that there is no influence of these aspect
in the presence of this chromosomal marker in cases of Chronic Myelogenous and
Acute Lymphoblastic Leukemias (CML and ALL, respectively) in Ecuador.
The study was performed in two major laboratories in Guayaquil (at the sea
level) and Quito (2800 m over the sea level) and included a population with
varied ethnical content. The described cases correspond to ALL and CML
diagnosed by bone marrow smears and Immunocytochemistry, this are 199 cases of
ALL and 295 cases of CML studied in both cities.
The CML presented and average frequency of Ph+ of 85%, and the ALL cases had a
frequency of 14%.
No statistical difference in the presence of Ph Chromosome in neither CML or
ALL was found in the studied cases at the coast area in relation to the
frequencies published for Quito and to the world statistics, although in a
previous publication of the molecular analysis of Quito’s cases it was shown
the presence of a particular pattern of the abl-bcr rearrangements.
P0093
Molecular and cytogenetic changes in STI571 (Gleevec) treated Acute
Lymphoblastic Ph + Leukemia
R. Kusec;
Cytogenetics Laboratory, Zagreb, CROATIA.
Targeting the tyrosine kines activity of Bcr-Abloncoprotein is effective
therapeuitc option of Ph-chromosome positive CML and ALL.
However, accumulating clinical experience describes emerging tumour resistance
to STI571 tyrosine kinase inhibitor in the treatment course.
A 36-year old woman with relapsing Philadelphia positive Acute Lymphoblastic
leukemia (Ph+ALL) was treated with STI571 (600 mg/d). After 3 months
haematological response in terms of correcting leukopenia, reducing the number
of immature cells in the bone marrow by 50% and decreasing the need for
platenet and haemoglobin transfusion was seen. However, in the sixth month of
treatment patient stopped responding to the drug with rapidly incresing number
of blasts. At that point standard G-banding of leukaemic cells identifed
additional chromosomal changes:del(6q) and t(11;14)(q13;q32). Molecurarly, we
were able to detect Major and Minor-breakpoint Bcr gene rearrangements in the
fusion with the Abl gene while molecular cytogenetics showed amplification of
the Bcr-Abl gene detected as the emergence of an extra Bcr-Abl gene copy in
17%of cells.PCR amplification of the BCL1-IgH fusion gene was negative and
there was no BCL1 expression by the blasts (immunocytochemistry). Biological
mechanisms of these genetic events are unknown and multiplication of
Philadelphia chromosome can be related to the acquired.
P0094
CGH In The Evaluation Of The Placenta In Abnormal Pregnancies
A. Amiel, N. Bouaron, R. Sharony, D. Kidron, M. Fejgin;
Meir Hospital, Kfar-Saba, ISRAEL.
Confined placental mosaicism (CPM) in term placental tissues is usually
diagnosed by conventional cytogenetic analysis and more recently by
fluorescence in situ hybridization (FISH) of the trophoblast. In this study,
we describe the use of comparative genomic hybridization (CGH) for detection
of chromosomal aneuploidy in 26 fresh and 14 paraffin embedded placentas and
evaluate the sensitivity of this novel approach for CPM diagnosis in multiple
placental samples.
We applied CGH technique to samples taken from various sites of placentas
originating from abnormal pregnancies (23 IUGRs, one with fetal malformation,
one with toxemia, one with hydrocephalus and 2 undetectable MSAFP). In the
control cases (7 normal and 5 with known aneuploidy) CGH concurred with the
known karyotype.
The most common aberration in the IUGR cases was the addition of a whole or
part of X chromosome. Other aberrations such as addition of Y chromosome ,
addition of 13(q22) and loss of chromosome 17 where found in other cases.
There was also one IUGR case of trisomy 8 (in one site) and 47,XXY found in
all sites. In the two cases with the MSAFP=O monosomy 16 was detected (in one
case on both sites searched). Some of the results were confirmed by the FISH
technique.
Our results demonstrate the usefulness of CGH technique in the genetic
evaluation of fresh and paraffin embedded placentas in problematic pregnancies
even when its morphology is normal.
P0095
Detection of chromosomal aneuploidy in spontaneous abortions using
comparative genomic hybridization (CGH)
N. V. Ostroverkhova, S. A. Nazarenko, I. N. Lebedev, A. D. Cheremnykh;
Institute of Medical Genetics, Tomsk, RUSSIAN FEDERATION.
Chromosomal aneuploidy is a common cause of abnormal prenatal development.
Comparative genomic hybridization (CGH) provides a rapid and comprehensive
detection chromosomal gains and losses in the test genome and maps the
aneuploidies onto normal metaphase chromosomes. Among the 52 tissue culture of
spontaneous abortions, 10 cases showed failure of fetal cell growth in culture
and could not be identified reliably by conventional cytogenetics. CGH
analysis was successfully performed for detection of chromosomal aneuploidy in
spontaneously aborted specimens with tissue culture failure. Balanced
karyotype profiles were obtained for 5 samples. All of them were analysed by
fluorescence in situ hybridization (FISH) with centromere-specific DNA probes
to exclude polyploidy. Nothing cells with abnormal level of ploidy were found.
Five spontaneous abortions (50%) have monosomy 22 and trisomy 10, 14, 18 and
21. Monosomy 22 identified by CGH is one of the most rare aneuploidies in
spontaneous abortions. In all cases with an indication of chromosomal
imbalance by CGH, FISH with chromosome-specific DNA probe was performed to
confirm the presence of aneuploidy. As determined by FISH analysis two cases
with trisomy 10 and monosomy 22 were mosaics with frequency of abnormal cell
line 68% and 33% respectively. Advantages and limitations of CGH for a
detection of complete and mosaic forms of aneuploidy are discussed.
P0096
Marker chromosome identification with chromosome microdissection and reverse
FISH and CGH
Y. H. Cho, J. Y. Lee, J. H. Kyhm, H. K. Seo, C. H. Lee;
Department of Medical Genetics, College of Medicine, Hanyang University, Seoul,
REPUBLIC OF KOREA.
Reverse painting fluorescent in situ hybridization (FISH) on the normal
metaphase with probes generated by chromosome microdissection and comparative
genomic hybridization (CGH) are powerful methods to identify the origin of
marker chromosomes. Three cases having unidentified marker chromosomes were
studied by reverse painting FISH and CGH. Reverse FISH probes were generated
from five copies of each marker chromosomes dissected with micromanipulator,
amplified with DOP-PCR, and labeled with fluorochromes. The probes were
hybridized to normal metaphases and the origin of marker chromosomes could be
determined. Three marker chromosomes were identified as derivative chromosome
15 inducing partial trisomy of 15q, duplication of the short arm of chromosome
17 and duplication of the short arm and deletion of the part of the long arm
of chromosome X. CGH showed concordant results with reverse FISH.
P0097
Reexamination of chromosome 2 rearrangements characterized by multicolor
banding (MCB) by region-specific FISH probes
A. Weise, H. Starke, A. Heller, U. Claussen, T. Liehr;
Institute of Human Genetics and Anthropology, Jena, GERMANY.
Conventional banding techniques often fail to characterize the exact nature of
chromosomal rearrangements. The MCB technique has demonstrated to improve the
definition of chromosomal breakpoints (e.g. Starke et al., 2001, PrenatDiag,
21, 1049-1052, Dufke et al., 2001, Europ J Hum Genet 9, 572-576). Here MCB was
applied to identify human chromosome 2 breakpoints in two clinical cases. To
show how precise the correlation between the MCB pseudocolors and the GTG
banding works the results of MCB were reexamined using region-specific YAC or
BAC probes. The chosen band resolution of chromosome 2 was 400 bands per
haploid karyotype. Case 1 presented with primary mental retardation and severe
delayed speech development. The boy had a der(9)t(2;9)(2q24.2;9p11.2)
according to GTG banding. MCB showed, however, that the translocation was
balanced although it seemed to be not according to GTG banding; new karyptype:
t(2;9)(q24.2;p24.3). Case 2 showed primary mental retardation, tendency to
seizures, craniofacial dysmorphisms and adipositas. The type of aberration in
this male patient could not be defined by GTG-banding
(inv(2)(p11q23)+dup?or.inv(2)(p21q24.1)+del?). MCB could characterize the
rearrangement as inv(2)(p15q24.3). In both cases the results of MCB were
confirmed with a panel of region specific YAC/BAC probes. In all 20 MCB
metaphases analyzed per case the breakpoints appeared within the same
pseudo-colored bands. Thus, the highly reproducible MCB pattern, can be used
to characterize abnormalities that remain cryptic or unresolvable in G-banding
analysis. Supported by DFG (436 RUS 17/40/00; PO284/6-1), Wilhelm
Sander-Stiftung (99.105.1), the EU (ICA2-CT-2000-10012 and QLRT-1999-31590).
Dr. Rocchi (Bari, Italy) is acknowledged for YAC/BACs.
P0098
Identification of satellite sequences in metaphase and interphase with
peptide nucleic acid (PNA) probes using multicolor fluorescence in situ
hybridization.
K. L. Taneja 1, B. Williams 1, R. H. Singer 2;
1Applied Biosystems, Bedford, MA, 2Albert Einstein College
of Medicine, Bronx, NY.
Multiplex fluorescence in situ hybridization (M-FISH) can be used to
detect marker chromosomes, chromosomal rearrangements in cancer, prenatal
diagnosis etc. Regular M-FISH requires a large amount of labeled DNA, the
hybridization time is longer and is less informative in interphase nuclei
compared to standard FISH. We have designed and developed directly labeled PNA
probes to distinguish up to 2 n-1 chromosomes (where n is the number
of different fluorochromes) using an epifluorescence microscope equipped with
a digital imaging camera and computer software for pseudocoloring and merging
images. Peptide nucleic acids (PNA) are synthetic mimics of DNA in which the
phosphodiester backbone has been replaced with 2-aminoethyl glycine linkages,
but maintaining the four natural nucleobases. PNA probes bind to the
complementary DNA sequence obeying Watson-Crick base pairing, however the
neutral backbone of the PNA molecule allows for the PNA/DNA binding to occur
more rapidly and more tightly than DNA/DNA binding. Chromosome specific
composite PNA probe sets were generated from the human satellite sequences, in
which the different fluorochromes were incorporated to address specific
issues, like identification of marker chromosomes and anueploidies. With four
fluorophores, we were able to enumerate up to 15 chromosomes in both metaphase
spreads and interphase nuclei in a single hybridization experiment. Our data
suggests that multiplex fluorescence in situ hybridization (M-FISH) using PNA
probes could have wide clinical utility, particularly in detection and
enumeration of chromosomes in a given sample. Multi-parameter hybridization
analysis should facilitate the study in molecular cytogenetics and probe-based
diagnosis of pathogens.
P0099
Multicolor fluorescent in situ hybridization in neuronal cells as an approach
for identification of low level chromosomal aneuploidy in the brain
Y. B. Yurov 1, V. M. Vostrikov 1, S. G. Vorsanova 2,
V. V. Monachov 1, I. Y. Iourov 1;
1National Center of Mental Health, Moscow, RUSSIAN FEDERATION, 2Intitute
of Pediatrics and Children Surgery, Moscow, RUSSIAN FEDERATION.
Fluorescence in situ hybridization (FISH) of DNA-DNA or DNA-RNA using
post-mortem brain samples is an approach to study a low-level chromosomal
aneuploidy and selective expression of specific genes in brain of patients
with neuropsychiatric diseases. We have performed a pilot
molecular-cytogenetic analysis of post-mortem brain of schizophrenic patients.
Multicolor FISH on two post-mortem brain samples of normal and six
schizophrenic individuals (area 10 of cortex) was applied. A set of DNA probes
for FISH included (i) centromeric alphoid DNA probes for chromosomes 7, 8, 13
and 21, 18, X, Y;(ii) classical satellite DNA probes for chromosomes 1 and 16
and (iii) region-specific DNA probes for chromosomes 13, 21 and 22.
Statistically significant level of aneuploidy (up to 3-4% of neurons)
involving chromosome X and 18 was detected in two post-mortem brains of
patients with schizophrenia. The multicolor FISH assay could be applied to
study low level of chromosomal aneuploidy, intranuclear distribution and
conformation of heterochromatin, abnormal patterns of chromosomal organization
and functional gene expression in situ in post-mortem brain at many
neurogenetic diseases. Schizophrenia and Rett syndrome are the diseases of
special interest for extended molecular-cytogenetic analysis as both of them
could suspect alterations in chromatin conformation and differential gene
expression in brain cells. Supported by Copernicus 2 grant.
P0100
CGH contribution in the delineation of chromosomal rearrangements
J. M. Lapierre, G. Joly, M. Prieur, O. Raoul, M. C. de Blois, N.
Morichon-Delvallez, P. Gosset, M. Vekemans, S. P. Romana, C. Turleau;
Hop.Necker-Enfants Malades, Paris, FRANCE.
Comparative Genomic Hybridization (CGH) is able to identify the origin of
extra or missing chromosome material when either the small size of the segment
or a non-discriminatory banding pattern does not allow a cytogenetic
diagnosis. It has also the potential to detect both terminal and interstitial
rearrangements. We illustrate here the contribution of CGH in the delineation
of 13 different cases studied in our laboratory. In most cases, CGH was
performed to characterize a rearrangement detected using classical cytogenetic
methods i.e identification of extra or missing chromosome material,
confirmation of an imbalance or accurate definition of the chromosome
breakpoints. In all these cases CGH was decisive. In some other cases,
classical cytogenetics (550 to 850 bands) did not detect any chromosome
imbalance when CGH detected a chromosome imbalance in several cases. All
abnormal results were confirmed using whole chromosome painting and/or FISH
with subtelomeric probes. In conclusion, CGH is a very powerful method to
analyze an unbalanced rearrangement already identified using classical
cytogenetics and requiring further characterization . When no chromosomal
rearrangement is observed, CGH could be an alternative to multiprobe FISH
study of all subtelomeric regions. However its use as a screening tool in
unexplained mental retardation remains limited due to the difficulty of
obtaining chromosomal preparations allowing high quality hybridizations on a
regular basis.
P0101
The Impact of BRCA1/2 susceptibility genes on women’s mental health
E. Dagan, S. Gil;
University of Haifa - Department of Nursing, Haifa, ISRAEL.
Three predominant mutations in BRCA1/2 genes have been found in 3% of the
Jewish Ashkenazi population. Such mutations significantly increase lifetime
risk for developing breast and/or ovarian cancer. The present study focuses on
the impact of being BRCA1/2 mutation carrier on women’s mental health. A
retrospective study was conducted in the oncogenetic clinic at Rambam medical
center, Israel. One hundred and thirty eight women were recruited and
evaluated regarding their medical history and mental health state using the
BSI (The Brief Symptom Inventory; Derogatise 1982). All women were genotyped
for BRCA1/2 founder mutations. Of the 138 women, 39 (28%) were mutation
carriers. Breast cancer was diagnosed in 69 (50%) women. The mean age at
diagnosis was 45.7±10.7 years and at the interview was 50±10.5 years.
Univariate analysis of Variance (ANOVA) [Morbidity (with/without breast
cancer) X Mutation (carrier/non-carrier)] revealed significant effect for
morbidity, mutation, and the interaction on four sub-scales of the BSI and on
its total score (GSI). Apparently, asymptomatic mutation carriers expressed
the highest levels of somatization (F=30.0; p<.001), depression (F=9.1;
p<.01), interpersonal sensitivity (F=4.5; p<.05) hostility (F=14.4;
p<.001), and GSI (F=8.9; p<.01). It may be that healthy women who carry
a mutation are more stressed regarding their health status than breast cancer
mutation carriers.
P0102
Familial or sporadic? Unexpected results in the diagnosis of hereditary
breast cancers
C. Schiffer, T. Voigtländer, R. Klaes;
Institute of Human Genetics, Heidelberg, GERMANY.
Genetic counseling and risk assessment in families with breast/ovarian cancer
is regularly based on pedigree analysis. However, familial and sporadic cases
may occur in the same family. We report on three families with unexpected
segregation of BRCA1/2 mutations in affected and unaffected family members.
Family No 1: The female proband, who presented with breast cancer at 28 years
of age, carried the common T300G mutation. Surprisingly her mother, diagnosed
with breast cancer at 33 years of age, was tested negative for this mutation.
T300G was identified in the proband´s healthy father.
Family No 2: Three siblings (one man, two women) and their deceased father had
been diagnosed with breast cancer. The brother and one sister, diagnosed at 40
years of age, carried the common 2041insA mutation in the BRCA2 gene. The
other sister, diagnosed at 60 years of age, tested negative for this mutation.
Family No 3: The female proband presented with breast cancer at 37 years of
age. She carried a novel BRCA1-splice mutation(4304+2insAdel21bp). Her mother,
diagnosed with breast cancer at 55 years, was tested negative, although she
had two affected sisters. The proband`s healthy father however with an
unremarkable family history carried the splice mutation. br />We conclude
that for exact risk assessment and genetic counseling in the hereditary
breast/ovarian cancer syndrome, each affected and unaffected family member at
risk should be tested.
P0103
Familial Dissemination of BRCA1/BRCA2 Test Results
J. C. Coyne 1, J. Stopfer 2, K. Calzone 1;
1University of Pennsylvania Health System, Philadelphia, PA, 2University
of Pennsylvania, Philadelphia, PA.
Study Design: Retrospective follow up study of individuals who notified that
they are carriers of a BRCA1/BRCA2 mutation. Participants received and
returned by mail an assessment of the pattern of their disclosure of the
results of their genetic testing in their family.
Instrumentation: Self-report questionnaire follow up assessment of mutation
carriers who have received results.
Most probands (60%) were the first in their family to receive results, and
almost half (47.3%) had agreements with family members prior to testing to
disclose results. Probands with such agreements were more likely to have
family members present during genetic counseling and results disclosure, C2
(1) = 4.0, p < .05. Individuals with such an agreement reported that a
sense of obligation, encouragement from their physician and family members,
and being asked by family members were stronger determinants of their decision
to share results than did probands without a prior agreement (all ps <
.001). Probands with such an agreement were more likely to endorse the
following factors as facilitating disclosure: support from close family
relationships, their physicians’ support, concern that family members be
able to use information to make healthcare decisions for themselves and their
children, and being asked directly by family members (all p < .05). These
data suggest that the family context is a crucial determinant of how genetic
testing information is disseminated, and that interventions aimed at improving
dissemination of genetic testing information need to focus on agreements to
disseminate test results made prior to the receipt of results.
P0104
Genetic polymorphisms of biotransformation enzymes and susceptibility to
breast cancer
J. Sarmanova 1, S. Susova 1, I. Gut 1,
J. Adamek 2, K. Kubackova 2, P. Soucek 1;
1National Institute of Public Health, Prague, CZECH REPUBLIC, 2Faculty
Hospital in Motol, Prague, CZECH REPUBLIC.
Breast cancer is the most common malignancy in women and second leading cause
of death from cancer. The genetically variable biotransformation enzymes:
epoxide hydrolase (EPHX), NADPH-quinone oxidoreductase (NQO1), and glutathione
S-transferases (GST's) metabolize drugs, carcinogens, and natural products. In
addition, it is generally accepted that majority of human cancers results from
exposure to environmental carcinogens. Considering the role in the metabolism
of chemicals played by biotransformation enzymes, we aimed at determining
whether any association exists between genetic polymorphisms of
biotransformation enzymes and individual susceptibility to breast cancer in
Czech women.
Genotyping analyses were performed by PCR-RFLP to determine the frequency of
polymorphisms in EPHX (exons 3 and 4), GSTM1 (deletion), GSTP1 (exon 5), GSTT1
(deletion) and NQO1 (exon 6). The study population consisted of 169 breast
cancer cases and 231 healthy controls.
No association between polymorphisms in EPHX, GSTM1, GSTP1, and GSTT1 and
breast cancer was found. On the contrary, a significantly different
distribution of genotypes in NQO1 between controls and breast cancer group was
confirmed by chi-square test (P=0.003, chi-square=11.83, DF=2). We have
observed significantly higher frequency of mutated genotype S/S in patients in
comparison with controls (8.1% vs. 1.3%). Homozygous mutant genotype S/S leads
to complete lack of activity NQO1. Moreover, the involvement of NQO1 in
colorectal cancer and tumor resistance to anticancer drugs was implicated.
Our results suggest that NQO1 may be an important factor in susceptibility to
breast cancer and its role should be further investigated.
This study was supported by grant GACR No.: 310/01/1537.
P0105
Mutational analysis of the Tuberous Sclerosis Complex (TSC) genes
N. D. Rendtorff 1, B. Mogensen 2, K.
Brondum-Nielsen 1, M. Schwartz 2;
1Department of Medical Genetics, The John F. Kennedy Institute,
Glostrup, DENMARK, 2Molecular Genetics Laboratory, Department of
Clinical Genetics, Rigshospitalet, Copenhagen, DENMARK.
Tuberous Sclerosis Complex (TSC) is an autosomal dominantly inherited disorder
characterized by development of benign tumours (hamartomas) in many organs.
Hamartoma formation in the central nervous system is associated with some of
the most problematic clinical manifestations of TSC, and can lead to
intellectual handicap, epilepsy and autism. Inactivating mutations in either
of two tumour supressor genes, TSC1 or TSC2, is the cause of this syndrome.
Here we have established a mutational analysis for TSC1 and TSC2. For the 21
coding exons of TSC1, we have developed a mutation identification assay that
combines long-range PCR with automated sequencing. For mutation screening of
the 41 coding exons of TSC2, we have developed a rapid and efficient
denaturing gradient gel electrophoresis (DGGE) assay.
We are currently collecting DNA samples from Danish tuberous sclerosis
patients. Presently, we are carrying out mutational analysis on DNA from
peripheral