ESHG Posters 2

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.

Y. Engwall¹, M. Nordling¹, J. Lundberg¹, A. Bergman¹, T. Martinsson¹, Z. Einbeigi², P. Karlsson², A. Wallgren², J. Wahlström¹;
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.

An investigation into the methylation status of oestrogen receptor beta and its subsequent expression in human breast cancer

F. J. Cooke¹, P. Balfe¹, A. H. McCann², P. Dervan³, M. Kennedy³, M. Kerin¹;
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 (ERb) 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 ERb promoter with respect to ERb expression by immunohistochemistry in a preliminary cohort of 25 primary human breast tumors. In addition we sought to correlate ERa, ERb 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 ERb 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.

M. Carvallo¹, L. Palma¹, M. Gallardo¹, C. Rousseau², M. King²;
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)

Telomerase enzyme activity and chromosome abnormalities detected by combined G-banding and comparative genomic hybridization in primary breast cancer

A. Papadopoulou¹, T. Trangas¹, H. Tsarouha¹, M. Texeira², E. Dimitriadis¹, P. Ioannidis¹, S. Heim³, N. Pandis¹;
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.

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.

A. Lyakhovich¹^,2, M. Shekhar¹;
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.

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.

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.

Androgen Receptor CAG Repeat Length in Jewish Israeli Women who are BRCA1/2 Mutation Carriers: Association with Breast/Ovarian Cancer Phenotype

E. Dagan¹, E. Friedman², T. Paperna¹, N. Carmi², R. Gershoni-Baruch¹;
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.

Association of 5382insC Mutation with SNPs of BRCA1 Gene and the Mutation Frequency in Russia

A. N. Loginova¹, N. I. Pospekhova¹, L. N. Lubchenko², E. V. Khomich¹, I. V. Kuzmina¹, A. V. Budilov³, V. M. Zakharyev³, E. K. Ginter¹, R. F. Garkavtseva², A. V. Karpukhin¹;
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.

BRCA2 mutations and polymorphisms in Russian patients with familial breast/ovarian cancer

E. V. Khomich¹, N. I. Pospekhova¹, L. N. Lubchenko², A. N. Loginova¹, I. V. Kuzmina¹, A. V. Budilov³, V. M. Zakharyev³, E. K. Ginter¹, R. F. Garkavtseva², A. V. Karpukhin¹;
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.

Prevalence of BRCA1 gene 5382insC mutation in St.Petersburg patients with familial breast cancer.

N. A. Grudinina¹, E. P. Lamber²;
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.

Altered expression of the candidate tumor suppressor gene, WWOX, in human breast tumors

K. Driouch¹, H. Prydz², R. Lidereau¹, E. Frengen²;
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).

Investigation of APC mutations of a patient with FAP and her family members by heterodublex analyses

B. Tunca¹, M. Menigatti², P. Benatti², G. Cecener¹, M. Pedroni², A. Scarselli², F. Borghi², E. Sala², T. Yýlmazlar³, A. Zorluoglu³, U. Egeli¹, O. Yerci⁴, M. Ponz de Leon²;
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.

Involvement of APC/beta-catenin signalling and E-cadherin in sporadic colon cancer

T. Cacev¹, R. Spaventi², K. Pavelic¹, S. Kapitanovic¹;
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.

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.

M. Kohoutová¹, J. Stekrová¹, V. Jirásek², J. Kotlas¹, V. Kebrdlová¹;
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.

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; 3949GàC 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.

S. Kapitanovic¹, T. Cacev¹, K. Pavelic¹, R. Spaventi¹^,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.

Complete characterization of the colon cancer cell line HT29 clone 19A by multicolor banding (MCB)

A. Kuechler¹^,2, A. Weise¹, S. Michel¹, B. Pool-Zobel³, A. Schaeferhenrich³, A. Heller¹, H. Starke¹, T. G. Wendt², U. Claussen¹, T. Liehr¹;
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).

Optimization of DHPLC experimental conditions for mutation analysis of the hereditary non polyposis colon cancer (HNPCC) genes hMLH1 and hMSH2

M. Schmitt¹, M. Gribba², J. M. Limacher³, S. Olschwang⁴, J. L. Mandel²;
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.

Unusual Findings Of APC Gene Analyses In suspected FAP Cases From The Republic Of Macedonia

A. J. Dimovski¹, A. M. Stefanovska¹, T. Josifovski², M. Panovski², D. Jashar³, G. Zografski³, G. D. Efremov¹;
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.

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.

Monozygotic twins showing variable expression of Muir-Torre syndrome due to MSH2 mutation.

S. R. Forrester¹, C. Baker², V. Kimonis³, M. Schneider¹;
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.

Mutations of N- and K-Ras, p53 and FMS genes in myelodysplastic syndromes in children

B. Jekic¹, V. Bunjevacki¹, M. Kuzmanovic², I. Novakovic¹, L. Lukovic¹;
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.

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. Egeli¹, T. Yakut¹, A. Bekar², M. Doygun², E. Ogul³;
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.

J. Mares¹, M. Babjuk², M. Trkova¹, J. Duskova³, V. Soukup², P. Goetz¹, Z. Sedlacek¹;
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.

Loss of heterozygosity of p53 gene in gastic carcinoma in the region of easten Turkey

I. Pirim¹, A. Karaman², M. Ikbal¹;
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.

P. Goetz¹, M. Trkova¹, L. Foretova², V. Krutilkova³, R. Kodet¹, J. Mares¹, Z. Sedlacek¹;
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.

Gene expression following tet-regulated reexpression of wt p53 in lung cancer cells

I. Wieland¹, A. Brüning², H. Burtscher³, U. H. Weidle³;
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.

GSTM1 null, GSTT1 null, GSTP1 (Ile105Val) and CYPA1 (T6235C) Genotypes in Childhood Acute Leukemia

G. Balta¹, E. Ozyurek¹, U. Ertem², G. Hicsonmez¹, C. Altay¹, A. Gurgey¹;
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.

Increased accuracy of leukemia diagnosis by combined analysis of morphology and FISH using the Duet automatic cell scanning system.

C. Kaplinski¹, I. Hardan¹, M. Daniely², A. Toren¹, A. Shimoni¹, A. Avigdor¹, M. Reichart², A. Nagler¹, T. Kaplan², F. Brok-Simoni¹, G. Rechavi¹, N. Amariglio¹, L. Trakhtenbrot¹;
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.

M. D. Tischkowitz¹, N. V. Morgan¹, C. Eddy¹, S. Ball², S. E. Langabeer³, I. Vorechovsky⁴, R. Stoeger¹, D. Grimwade¹^,3, C. G. Mathew¹, S. V. Hodgson¹;
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*

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.

The relationship between the chromosomal rearrangement complexity and disease agresivity in some cases of leukemia

A. G. Lungeanu¹, A. Arghir², A. Lupu³, D. Mut-Popescu⁴, N. Berbec⁴, L. Popescu⁵;
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.

D. Szczesniak¹, J. Kocki¹, M. Cioch², A. Dmoszynska², B. Marzec¹, J. Wojcierowski¹;
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.

Submicroscopic deletion at the breakpoint in chromosome der(9) in Ph+ acute lymphoblastic leukemia (ALL)

I. F. Loncarevic-Barcena¹, B. Shell¹, H. J. Fricke², M. Prechtel¹, M. Ziegler¹, U. Claussen¹;
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.

Acute monocytic leukemia and multiple abnormalities in a child with duplication of 1q detected by GTG-banding and SKY.

M. R. Baruffi¹, C. A. Scrideli², J. A. Squire³, J. Karaskowa³, E. S. Ramos⁴, B. Heck⁴, L. G. Tone⁴;
¹Ribeirao Preto Medice School, University of Sao Paulo, Ribeirao Preto, SP, BRAZIL, ²Ribeirao Preto Medicine School, University of Sao Paulo, Ribeirão 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

A. Carrió¹, D. Costa¹, A. Arias¹, R. Queralt¹, F. Cervantes², J. Aguilar³, D. Colomer³, E. Campo³;
¹Servei de Genètica. 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.

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.

M. Alkan¹, C. Ulger¹, G. A. Toruner¹, M. Muhammed², S. Damani², P. Tolias¹^,3, M. Schwalb¹, J. Dermody¹;
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.

High-throughput tissue microarray analysis of 11q13 genes amplification (CCND1, FGF3/FGF4, FGF3, EMS1) in urinary bladder cancer

B. Zaharieva¹, R. Simon², T. Gasser³, G. Sauter², D. Toncheva¹;
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.

Dystrophic Scoliosis And Genetic Polymorphisms In Patients With Neurofibromatosis

S. Funke¹, E. Morava², M. Czakó³, B. Cser⁴, G. Kosztolányi⁴, T. Illés⁵;
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.

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.

Do some additional chromosome rearrangements mean a favourable T-cell prolymphocytic leukemia prognosis with preserved alkylator based treatment sensitivity?

N. Kokalj Vokac¹, S. Zver², A. Erjavec¹, B. Zagradisnik¹, A. Zagorac¹, D. Zontar², P. Cernelc²;
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.

Prognostic Significance Of Small Cell Clones With Hyperdiploidy In Childhood Acute Lymphoblastic Leukemia (all).

Z. Zemanova¹, K. Michalová¹^,2, L. Sindelarova¹, J. Brezinova², S. Kurkova², P. Smisek³, O. Hrusak⁴, J. Stary³;
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.

Cytogenetic studies in T-cell acute lymphoblastic leukemia: a report of 35 cases.

N. Douet-Guilbert¹, M. J. Le Bris², A. Herry², F. Morel², G. Le Calvez³, V. Marion³, J. F. Abgrall³, C. Berthou¹, M. De Braekeleer²;
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.

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.

Molecular and cytogenetic changes in STI571 (Gleevec) treated Acute Lymphoblastic Ph + Leukemia

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.

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.

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.

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.

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.

Identification of satellite sequences in metaphase and interphase with peptide nucleic acid (PNA) probes using multicolor fluorescence in situ hybridization.

K. L. Taneja¹, B. Williams¹, R. H. Singer²;
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 2ⁿ-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.

Multicolor fluorescent in situ hybridization in neuronal cells as an approach for identification of low level chromosomal aneuploidy in the brain

Y. B. Yurov¹, V. M. Vostrikov¹, S. G. Vorsanova², V. V. Monachov¹, I. Y. Iourov¹;
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.

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.

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.

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.

J. C. Coyne¹, J. Stopfer², K. Calzone¹;
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.

Genetic polymorphisms of biotransformation enzymes and susceptibility to breast cancer

J. Sarmanova¹, S. Susova¹, I. Gut¹, J. Adamek², K. Kubackova², P. Soucek¹;
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.

N. D. Rendtorff¹, B. Mogensen², K. Brondum-Nielsen¹, M. Schwartz²;
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