Complementation of non-tumorigenicity of HPV18-positive cervical carcinoma cells involves differential mRNA expression of cellular genes including potential tumor suppressor genes on chromosome 11q13.

The fusion between human tumorigenic cells and normal human diploid fibroblasts results in non-tumorigenic hybrid cells, suggesting a dominant role for tumor suppressor genes in the generated hybrid cells. After long-term cultivation in vitro, tumorigenic segregants may arise. The loss of tumor suppressor genes on chromosome 11q13 has been postulated to be involved in the induction of the tumorigenic phenotype of human papillomavirus (HPV)18-positive cervical carcinoma cells and their derived tumorigenic hybrid cells after subcutaneous injection in immunocompromised mice. The aim of this study was the identification of novel cellular genes that may contribute to the suppression of the tumorigenic phenotype of non-tumorigenic hybrid cells in vivo. We used cDNA microarray technology to identify differentially expressed cellular genes in tumorigenic HPV18-positive hybrid and parental HeLa cells compared to non-tumorigenic HPV18-positive hybrid cells. We detected several as yet unknown cellular genes that play a role in cell differentiation, cell cycle progression, cell-cell communication, metastasis formation, angiogenesis, antigen presentation, and immune response. Apart from the known differentially expressed genes on 11q13 (e.g., phosphofurin acidic cluster sorting protein 1 (PACS1) and FOS ligand 1 (FOSL1 or Fra-1)), we detected novel differentially expressed cellular genes located within the tumor suppressor gene region (e.g., EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) and leucine rich repeat containing 32 (LRRC32) (also known as glycoprotein-A repetitions predominant (GARP)) that may have potential tumor suppressor functions in this model system of non-tumorigenic and tumorigenic HeLa x fibroblast hybrid cells.

Silver-Russell syndrome: genetic basis and molecular genetic testing.

Imprinted genes with a parent-of-origin specific expression are involved in various aspects of growth that are rooted in the prenatal period. Therefore it is predictable that many of the so far known congenital imprinting disorders (IDs) are clinically characterised by growth disturbances. A noteable imprinting disorder is Silver-Russell syndrome (SRS), a congenital disease characterised by intrauterine and postnatal growth retardation, relative macrocephaly, a typical triangular face, asymmetry and further less characteristic features. However, the clinical spectrum is broad and the clinical diagnosis often subjective. Genetic and epigenetic disturbances can meanwhile be detected in approximately 50% of patients with typical SRS features. Nearly one tenth of patients carry a maternal uniparental disomy of chromosome 7 (UPD(7)mat), more than 38% show a hypomethylation in the imprinting control region 1 in 11p15. More than 1% of patients show (sub)microscopic chromosomal aberrations. Interestingly, in approximately 7% of 11p15 hypomethylation carriers, demethylation of other imprinted loci can be detected. Clinically, these patients do not differ from those with isolated 11p15 hypomethylation whereas the UPD(7)mat patients generally show a milder phenotype. However, an unambiguous (epi)genotype-phenotype correlation can not be delineated.We therefore suggest a diagnostic algorithm focused on the 11p15 hypomethylation, UPD(7)mat and cryptic chromosomal imbalances for patients with typical SRS phenotype, but also with milder clinical signs only reminiscent for the disease.

Amplicons on human chromosome 11q are located in the early/late-switch regions of replication timing.

Amplicons are frequently found in human tumor genomes, but the mechanism of their generation is still poorly understood. We previously measured the replication timing of the genes along the entire length of human chromosomes 11q and 21q and found that many "disease-related" genes are located in timing-transition regions. In this study, further scrutiny of the updated replication-timing map of human chromosome 11q revealed that both amplicons on human chromosomal bands 11q13 and 11q22 are located in the early/late-switch regions of replication timing in two human cell lines (THP-1 and Jurkat). Moreover, examination of synteny in the human and mouse genomes revealed that synteny breakage in both genomes occurred primarily at the early/late-switch regions of replication timing that we had identified. In conclusion, we found that the early/late-switch regions of replication timing coincided with "unstable" regions of the genome.

Quantifying chromosome changes and lineage involvement in myelodysplastic syndrome (MDS) using fluorescent in situ hybridization (FISH).

A simplified technique for fluorescent in situ hybridization (FISH) was used to investigate the prevalence of chromosomally abnormal clones in 13 cases of myelodysplastic syndrome (MDS). Biotinylated centromeric probes for chromosomes 7, 8, 12 and X, as well as painting probes for chromosomes 7 and 11, were applied to air-dried bone marrow smears stored from 6 to 23 months. Nine of the cases had been previously karyotyped, and five of these demonstrated normal karyotypes which were confirmed by FISH. The remaining four cases showed different chromosome changes. One case of sideroblastic anemia with chronic lymphocytic leukemia showed minor clones with either monosomy 12 (12% of cells) or tetraploidy (15% of cells) by FISH, whereas metaphase cytogenetics had demonstrated trisomy 12 in 20% of cells, with no evidence of tetraploidy. Another case which had been previously karyotyped was found to have a t(7;11) in 90% of cells while only 10% of cells were shown by FISH to contain this translocation. Monosomy 7 was demonstrated by FISH in a case of refractory anemia (RA), while trisomy 8 was found in a case of RA with excess blasts in transformation (RAEB-T), and in both of these cases the aneuploid clone was present in eosinophils as well as in erythroid and granulocytic precursors but not in lymphocytes or histiocytes, thereby demonstrating the value of FISH for identifying the affected cell lineage.

Translocation t(1;22) mimicking t(1;19) in a child with acute lymphoblastic leukemia as revealed by chromosome painting.

The karyotype of a boy with acute lymphoblastic leukemia (ALL) presenting with numerical and structural chromosome aberrations as determined by Giemsa-banding was further investigated using chromosome painting (CP). A translocation t(11;18)(q23;q21) was verified by this approach, and gain of chromosome 21 material due to a structural rearrangement was detected. Moreover, an unbalanced translocation of the long arm of chromosome 1, resembling the well known translocation t(1;19), was demonstrated to involve chromosome 22 instead of chromosome 19. Immunophenotyping of the leukemic blasts led to the diagnosis common ALL (CD19+, CD10+, clg-). Our case indicates that in ALL a translocation t(1;19) may be mimicked by other chromosomal rearrangements, and that CP may efficiently complement conventional cytogenetics in the exact characterization of the involved chromosomes.

Decrease in catalase activity and loss of the 11p chromosome arm in the course of SV40 transformation of human fibroblasts.

The activity of catalase, a key enzyme in cell detoxication of oxygen derivatives, was studied in SV40 transformed human fibroblasts. A cytogenetic study was performed in parallel to establish a quantification of 11p arm on which the corresponding gene is mapped. mRNA amounts were determined by Northern blotting. At early passages, catalase activity strongly decreased whereas the corresponding mRNA was present. No deletions of 11p arms were detected. At later passages, catalase activity remained low. 11p arm deletions were frequent, and the amount of mRNA was decreased. In these late passages, the good correlation between the number of 11p arms and catalase activity suggested a gene dosage effect. It is assumed that the decrease of catalase activity provides a selective advantage for the transformed cells. This decrease is related to a post-transcriptional change of regulation at early passages and to the loss of the corresponding gene at later passages.

Common region of ALL-1 gene disrupted in epipodophyllotoxin-related secondary acute myeloid leukemia.

Translocations at chromosomal band 11q23 characterize most de novo acute lymphoblastic leukemias (ALL) of infants, acute myeloid leukemias (AML) of infants and young children, and secondary AMLs following epipodophyllotoxin exposure. The chromosomal breakpoints at 11q23 have been cloned from isolated cases of de novo ALL and AML. Using an 859-base pair BamHI fragment of human ALL-1 complementary DNA that recognizes the genomic breakpoint region for de novo ALL and AML, we investigated two cases of secondary AML that followed etoposide-treated primary B-lineage ALL. In the first case, the translocation occurred between chromosomes 9 and 11 and the breakpoint at 11q23 localized to the same 9-kilobase region of the ALL-1 gene that is disrupted in most of the de novo leukemias. In the second case the translocation was between chromosomes 11 and 19. The breakpoint occurred outside of the ALL-1 breakpoint cluster region.

Loss of heterozygosity at 11p13 in Wilms' tumours does not necessarily involve mutations in the WT1 gene.

Loss of heterozygosity (LOH) in tumour cells is generally accepted as 'exposing' recessive cancer genes. The short arm of chromosome 11 shows consistent LOH in Wilms' tumours along its entire length. Occasionally, however, only the 11p13 and/or the 11p15 regions are involved. Deletions of the 11p13 region consistently predisposes to Wilms' tumorigenesis. We have analysed the recently cloned WT1 gene from the 11p13 region exon-by-exon in five tumours previously shown to have undergone LOH for the 11p13 region, using single strand conformation polymorphism analysis (SSCP) and PCR sequencing. Our analysis using SSCP failed to identify any band shifts in the WT1 gene from these tumours. In addition we also sequenced the zinc finger region of WT1, which is the part of the gene most frequently showing mutations. Only the normal sequence was found in all of these tumours. These results demonstrate that LOH in Wilms' tumours is not always related to mutations in the WT1 genes and argues strongly that another gene, probably in the 11p15 region, may be more important in Wilms' tumorigenesis.

Exclusion of linkage between schizophrenia and the D2 dopamine receptor gene region of chromosome 11q in 112 Irish multiplex families.

A leading theory hypothesizes that schizophrenia arises from dysregulation of the dopamine system in certain brain regions. As this dysregulation could arise from abnormal expression of D2 dopamine receptors, the D2 receptor gene (DRD2) on chromosome 11q is a candidate locus for schizophrenia. We tested whether allelic variation at DRD2 and five surrounding loci cosegregated with schizophrenia in 112 small- to moderate-size Irish families containing two or more members affected with schizophrenia or schizoaffective disorder, defined by DSM-III-R. Evidence of linkage was assessed using varying definitions of illness and modes of transmission. Assuming genetic homogeneity, linkage between schizophrenia and large regions of 11q around DRD2 could be strongly excluded. Assuming genetic heterogeneity, variation at the DRD2 locus could be rejected as a major risk factor for schizophrenia in more than 50% of these families for all models tested and in as few as 25% of the families for certain models. The DRD2 linkage in fewer than 25% of these families could not be excluded under any of the models tested. Our results suggest that the major component of genetic susceptibility to schizophrenia is not due to allelic variation at the DRD2 locus or other genes in the surrounding chromosomal region.

A linkage study of chromosome 11q in schizophrenia.

The long arm of chromosome 11 is of interest in schizophrenia research because of three independent reports of balanced 11q translocations cosegregating with schizophrenia and other major psychiatric illness in pedigrees. In addition, a number of candidate genes for psychosis are located in the translocated regions. These include the dopamine D2 receptor, porphobilinogen deaminase, which has shown an allelic association with schizophrenia, and neural cell adhesion molecule, a cell surface glycoprotein involved in neuronal cell-cell recognition during brain development. To search for a schizophrenia locus on chromosome 11q, we conducted linkage analyses in 12 multiplex pedigrees. Sixteen DNA markers, including the above three candidate genes, were used to screen the entire long arm of chromosome 11. None of these markers were supportive of linkage to schizophrenia regardless of whether the affected phenotype was defined narrowly or broadly, whether high or low penetrance was assumed. Both dominant and recessive models tested more than 130 centimorgans of chromosome 11q, and therefore, the reported translocation regions. The results provide no evidence for a susceptibility locus for schizophrenia on chromosome 11q in these pedigrees.

Loss of heterozygosity and amplification on chromosome 11q in human ovarian cancer.

The 11q13 chromosomal region encodes oncogenes relevant to a variety of human cancers as well as a tumour suppressor gene implicated in multiple endocrine neoplasia type 1. In addition, high affinity folate receptor (FOLR1), which maps to 11q13.3-13.5, is expressed at an elevated level on the surface of over 80% of nonmucinous epithelial ovarian cancers. Further telomeric, 11q breakpoints are found in many cancers. We studied the involvement of 11q markers in ovarian cancer by looking for tumour-specific loss of heterozygosity (LOH), as well as amplification or rearrangements that might explain the overexpression of FOLR1. Twenty eight epithelial ovarian cancers, along with lymphocyte DNA from the same individual were used for Southern blotting with polymorphic probes from 11q. PCR primers from 11q23.3 were also used. The 11q13 band was amplified in four out of 28 cancers. The amplicon included the probe D11S146 as well as FGF3 (formerly INT2) and FOLR1 in one out of these four cases, thus crossing the bcl1 translocation breakpoint. LOH was seen in three out of 16 cases with FGF3 (11q13). A much higher frequency of LOH (8/12) was found at 11q23.3-qter, implying the presence of a tumour suppressor gene in this region.

Regulation of human papillomavirus type 16 (HPV-16) transcription by loci on the short arm of chromosome 11 is mediated by the TATAAAA motif of the HPV-16 promoter.

The human papillomavirus type 16 (HPV-16) enhancer-promoter is virtually inactive in normal human diploid fibroblasts, but active in human fibroblasts with a deletion in the short arm of one chromosome 11 (del-11 cells). Since the HPV-16 enhancer with the simian virus 40 promoter is active in both cell types, the target for chromosome 11-regulated HPV-expression is likely to be located in the HPV-16 early promoter region (nucleotides 57 to 112). We show here that DNA-protein complexes formed with an HPV-16 promoter fragment are quantitatively different in del-11 cell and diploid cell extracts. This quantitative difference detected in band shift experiments disappeared upon mutation of the HPV-16 TATAAAA box to TATTTAT. This mutation also strongly reduced the activity of the HPV-16 enhancer-promoter in del-11 cells. These results indicate that TATA-binding proteins are involved in the chromosome 11-mediated regulation of HPV-16 gene expression.

Diagnosis of Ewing's sarcoma and peripheral neuroectodermal tumour based on the detection of t(11;22) using fluorescence in situ hybridisation.

Fluorescence in situ hybridisation (FISH) has been used increasingly for gene mapping and ordering probes on interphase and metaphase preparations. The association of consistent chromosomal aberrations with certain malignancies allows the possibility of using interphase cytogenetics as a diagnostic tool. In small round cell tumours of children accurate diagnosis may be difficult using existing methods. We have therefore evaluated the diagnostic potential of this technique when applied to the characteristic t(11;22) found in Ewing's sarcoma and peripheral neuroectodermal tumour (ES and PNET). Interphase nuclei were prepared from normal human foreskin fibroblasts (HFF), two Ewing's sarcoma cell lines and several fresh tumour biopsies. DNA probes each side of the breakpoint at 22q12 were labelled with biotin and digoxygenin, hybridised to chromosomes in interphase and detected in different colours. Measurements between pairs of signals arising from each copy of chromosome 22 were taken and statistical analysis performed. There was a highly significant difference (P < 0.0001) between the two populations of measurements obtained (from nuclei with and without the t(11;22)). Studying four tumours and one further ES line (blind) it was found that median values from 30 nuclei could correctly identify which samples contained the t(11;22). This application of interphase cytogenetics contributes a reliable, accurate and conceptually simple diagnostic test for ES and PNET. It may now be applied to other tumours with characteristic translocations, amplifications or deletions when suitable probes are available. This approach is likely to become a routine in clinical diagnosis.

Definition of a small chromosomal rearrangement on chromosome 11p14 by molecular cytogenetics in situ hybridization.

Chromosomal in situ hybridization allows the detection and the definition of single copy DNA segments of very small size. In a particular case, we demonstrate the inactivity of this molecular cytogenetic technique. In this case, karyotype analysis revealed a chromosome 11p+. In situ hybridization of probes PBGD, D11S29, NCAM, and ETSI located at 11q23-qter shows that the extra chromosomal material on chromosome 11p+ is a duplication of the 11q23-qter region.

Chrysotile fiber is a strong mutagen in mammalian cells.

Although chrysotile asbestos is a proven human carcinogen, several studies have concluded that these fibers are not mutagenic to cultured mammalian cells. We show here, on the other hand, that when tested using the AL cell system that detects both intragenic and multilocus mutations, chrysotile is indeed mutagenic and comparable in strength to that of gamma-rays. Southern analysis of the induced mutants shows that the majority contains large deletions ranging in size from a few thousand to several million base pairs. Results of our study demonstrate that, while chrysotile may be less durable in vivo than the amphibole fibers such as crocidolites and amosites, it can effectively create genetic damage involved in the cancer process.

Clonal rearrangement of chromosome band 6p21 in the mesenchymal component of pulmonary chondroid hamartoma.

Pulmonary chondroid hamartomas (PCH) are biphasic benign tumors that contain both mesenchymal and epithelial populations. In this report we describe two PCH in which clonal translocations at chromosome band 6p21 were demonstrated in mesenchymal cells. One of these had a unique translocation, t(6;14)(p21;q24), that was also found in one of two PCH karyotyped previously. The t(6;14) has not been described in other varieties of benign or malignant neoplasia. The 6p21 aberrations are of particular interest because break points in this chromosomal region appear to be characteristic of endometrial polyps. Endometrial polyps, like PCH, are biphasic benign tumors in which mesenchymal clonality has been demonstrated.

Amplification of genes within the chromosome 11q13 region is indicative of poor prognosis in patients with operable breast cancer.

Amplification of the chromosome 11q13 region, which harbors the BCL1 region and the PRAD1, EMS1, HSTF1, and INT2 genes, was found in 36 (16%) of a series of 226 breast carcinomas. In the 153 patients with stage I-IIIa disease who had received no therapy prior to surgery and who were treated with curative intent, 11q13 amplification was associated with the presence of lymph node metastases (P less than 0.002). The presence of an 11q13 amplification was associated with a significantly shorter relapse-free survival (P less than 0.002) and a higher breast cancer-specific mortality (P less than 0.003). Stepwise multivariate analysis showed that, in addition to lymph node status, 11q13 amplification was the best predictor for short survival. Stratified log-rank analysis indicated that, within the group of lymph node-positive breast cancer patients, 11q13 amplification identifies a subgroup at high risk.

Aniridia--Wilms' tumour association--a case with 11p 13-14.1 deletion and ventricular septal defect.

A two year old female child with bilateral wilms tumor (WT) along with multiple congenital anomalies like bilateral aniridia with congenital cataracts and nystagmus, microcephaly, mental retardation and ventricular septal defect has been described. The karyotype analysis revealed 46 xx, del 11p 13-14.1. Association of ventricular septal defect with the classical features of 'Aniridia-Wilms' tumor association' is an unusual feature in this case.

Association between chromosome 11q13 amplification and prognosis of patients with oesophageal carcinomas.

Ninety pairs of normal and tumour tissue DNAs were isolated from paraffin-embedded blocks of advanced oesophageal carcinoma cases and examined for gene amplification at chromosome 11q13 by dot-blot hybridisation using the int-2 gene as a probe. 22 of 90 carcinomas (24%) showed more than two times amplification. Although no significant correlation was observed between gene amplification and histological type or metastasis to lymph node, a tendency for deeper invasion to be associated with more frequent amplification was observed. In relation to prognosis, patients with amplification had a lower survival rate than those without amplification. This tendency was evident both in the group with well differentiated type carcinoma and in the group which had no metastasis to lymph node. Thus, gene amplification of the int-2 locus may be a useful prognostic factor.