Biomedical subjects

A Bernheim

Publications and source records attributed to A Bernheim.

At least 19 recordsLinked to original sources

PubMed1996

Integrated physical, genetic, and genic map covering 3 Mb around the human NGF gene (NGFB) at 1p13.

We have characterized 11 overlapping yeast artificial chromosomes (YACs) in the 1p13 region, 8 of them containing the human nerve growth factor (NGF) gene (HGMW-approved symbol NGFB). Sequence-tagged sites (STSs) corresponding to YAC extremities have been designed and used for chromosome assignment on a panel of monochromosomic somatic cell hybrids to check for YAC chimerism, in parallel with analyses by fluorescence in situ hybridization. Determination of end STS content and restriction mapping of the YACs led to the construction of a 3-Mb YAC contig. Four microsatellite markers from the Généthon collection and seven genes known to map to the 1p13 region have been ordered on the contig around the NGF gene. A new gene transcript from the Genexpress catalog has been localized on the contig. This work provides an integrated physical, genetic, and genic map of this chromosome 1 region. It constitutes a basis for determining the structure of the NGF gene and for further characterizing its genic environment.

Animals↗

PubMed1996

High incidence of the t(2;5)(p23;q35) translocation in anaplastic large cell lymphoma and its lack of detection in Hodgkin's disease. Comparison of cytogenetic analysis, reverse transcriptase-polymerase chain reaction, and P-80 immunostaining.

Fifty-six cases of anaplastic large cell lymphoma (ALCL), 23 cases of Hodgkin's disease, and 16 cases of diffuse large cell lymphoma were investigated for the t(2;5)(p23;q35) translocation. The translocation was detected by using cytogenetic analysis, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry with P80 antibody directed against the kinase domain of anaplastic lymphoma kinase (ALK) of the chimeric NPM/ALK protein. In all but three cases of ALCL, we found an agreement between cytogenetic analysis, RT-PCR, and P80 staining. However, in one case, the t(2;5) translocation was detected with cytogenetic analysis, but RT-PCR and P80 staining were found to be negative. Conversely, in another case the karyotype was normal, but the hybrid mRNA and P80 staining were found to be positive. In one case, malignant cells showed a translocation involving chromosomes 1q25 and 2p23 and were strongly positive for P80 staining. Such a result could be expected because P80 antibody detects the kinase domaine of the ALK protein encoded by chromosome 2p23. Overall 73.2% (41 of 56) of cases were found to be positive. However, the highest percentage (23 of 26 cases; 88.5%) of P80 positive cases was found in children compared with 60% (18 of 30 cases) in adult ALCL (P < .05). In Hodgkin's disease, Reed-Sternberg cells were found to be clearly negative by RT-PCR and with P80 antibody. The latter results suggest that Hodgkin's disease and t(2;5)-positive ALCL are distinct biological entities and that the demonstration of the t(2;5) translocation is of diagnostic importance in differentiating these two entities. The results of the present study indicate that immunohistochemistry with P80 antibody is a reliable method for detecting NPM/ALK chimeric protein.

Adolescent↗

PubMed1996

Interphase cytogenetic studies of human hepatocellular carcinomas by fluorescent in situ hybridization.

Although numerous allelic chromosome losses have been reported in hepatocellular carcinomas (HCC), chromosome analysis by cytogenetic methods has rarely been performed in these tumors, unlike other solid malignant tumors. The purpose of the current study was to analyze primary liver tumors by conventional cytogenetic methods and by a new molecular cytogenetic technique, called fluorescent in situ hybridization (FISH), a technique that has been recently proposed to count the number of chromosome copies in interphase nuclei with chromosome centromeric probes. Primary cultures of tumoral cells were prepared to obtain metaphases. Specific chromosomes probes 7, 17, and 20 were used to perform in situ hybridization on isolated intact tumoral cells. Seven cases of primary liver tumors (six cases of HCC and one case of benign focal hepatic nodular hyperplasia) were investigated. A few metaphases were obtained in five of the seven tumors, and in most cases numerical abnormalities were difficult to interpret. In contrast with in situ hybridization, all cases of HCC showed losses and/or gains of chromosomes. Loss of one to three chromosomes occurred in five tumors. A gain of two chromosomes was observed in two of these five tumors. In only one case, a gain of only three chromosomes occurred. In addition, a loss of chromosome 17 was recorded for the benign tumor. These results demonstrate that FISH with specific probes can provide information on chromosome number in the tumoral cells of primary liver tumors even in the absence of analyzable metaphases. This technique opens new possibilities for the investigation of chromosome abnormalities in HCC.

Adult↗

PubMed1996

Assignment of the NTRK4 (trkE) gene to chromosome 6p21.

Reverse transcriptase-polymerase chain reactions using foetal brain RNA with reverse and forward primers of the first, second and third NTRK4 region allowed us to obtain three amplified NTRK4 fragments. The specificity of amplified fragments was checked by digestion with restriction endonucleases AvrII, HindIII and PspII for the first, second and third regions, respectively. Each restriction site was specific for each amplified fragment. The fragment of the NTRK4 first region was also sequenced and the sequence determined was identical to the human NTRK4 sequence. The three amplified fragments were cloned in pBS. For the Southern technique, plasmid pBS-NTRK4a (with an insert of 1052 bp) detected a human 9-kb HindIII sequence which was localised unambiguously on chromosome 6. For fluorescence in situ hybridisation, the three plasmids, pBS-NTRK4a, pBS-NTRK4b (insert 924 bp) and pBS-NTRK4c (insert 1114 bp) were pooled and used as a probe. This NTRK4 probe was localised on 6p21. Of 50 metaphases analysed, 49 contained twin spot signals on both sister chromatids.

Base Sequence↗

PubMed1996

MDM2 amplification in a primary alveolar rhabdomyosarcoma displaying a t(2;13)(q35;q14).

This report describes a case of rhabdomyosarcoma associated with a 2;13 translocation and multiple double minute chromosomes. The origin of the amplified DNA was identified using comparative genomic hybridization, which pinpointed a unique spot at 12q13-->q14. Band 12q13 has been shown to contain several genes that are occasionally amplified in other sarcomas. Fluorescene in situ hybridization to tumor metaphases with probes specific for this region indicated that the double minutes contained the MDM2 gene but not the CDK4 gene. MDM2 amplification was further quantified by Southern hybridization, which showed a mean value of 25 copies per haploid genome. This is the first example of MDM2 amplification in a rhabdomyosarcoma.

Adolescent↗

PubMed1996

Three-dimensional fluorescence in situ hybridization. Chromosomal studies on nuclei from cytogenetic preparations by confocal microscopy.

OBJECTIVE: To determine the three-dimensional (3D) position of target sequences and chromosomal volumes in interphase human nuclei by confocal laser scanning microscopy (CLSM) by using the heterochromatin part of the long arm of human chromosome Y (HPLAHC) as a target for specific DYZ1 probes, then by D10Z1 probes specific to the centromere of chromosome 10. STUDY DESIGN: Fluorescence in situ hybridization information inside chromosomal preparations was obtained with FITC-labelled probes and propidium iodide (PI) as a DNA-specific stain. To have a control in the experiment, HPLAHC Y was taken as a model of a domain and the centromere of chromosome 10 as a model of a single centromere spot. To have access to their 3D visualization, we selected FITC and PI patterns of fluorescence when optical slices were obtained and used a 3D reconstruction software. RESULTS: Labelling of the target by the probes was characteristic of Y heterochromatin and chromosome 10 centromere localizations and allowed observation of their domain in the x, y and z directions. CONCLUSION: This work was performed on two sets of 30 stained interphase nuclei. Deformations were confirmed by fluorescent spherical beads mounted in the same medium and scanned in the same conditions.

Cell Nucleus↗

PubMed1996

Mapping of 22 YACs on human chromosomes by fish using yeast DNA Alu-PCR products for competition.

Chromosomal assignment and analysis of chimerism of 22 YACs was performed by FISH. Probes were obtained by PCR amplification of the human YAC inserts with Alu primers. Maximum amplification of various inter-Alu elements was obtained when the primer annealing temperature was below the optimal temperature needed for high specificity. In these conditions, yeast DNA contributed to the amplification of various Alu-PCR products and, since strong competition was required for the suppression of all Alu sequences, yeast Alu-PCR products fulfilled this purpose efficiently.

Base Sequence↗

PubMed1996

Cytogenetic analysis of BC2, a new human hepatoma cell line, by fluorescent in situ hybridization.

Cytogenetic analysis of a new human hepatoma cell line BC2 was performed with conventional cytogenetic techniques and fluorescent in situ hybridization. Numerical and structural abnormalities were observed by conventional cytogenetics for chromosomes 1, 2, 4, 7, 8, 9, 10, 11, 15, 17 and 20. Chromosome painting allowed to specify the translocation of chromosome 1, and to characterize 3 markers from chromosome 8 and one marker from 9, which were unrecognizable by conventional techniques. Comparison of chromosome 1 abnormalities with those reported in the literature for other human hepatoma cell lines showed that structural abnormalities of chromosome 1 were present in different regions of this chromosome. A review of the literature was done, and the results discussed, suggesting that alterations of chromosome 1 may be important in hepatocarcinogenesis.

Carcinoma, Hepatocellular↗

PubMed1995

Gonadal dysgenesis in del(18p) syndrome.

We report on a girl with syndromal gonadal dysgenesis and a de novo del(18p). Genetic factors controlling gonadal development are located not only on the X chromosome, but also on autosomes. The present case suggests that one of these genes is situated on 18p. We conclude that patients with del(18p) syndrome should be evaluated for gonadal dysgenesis.

Child, Preschool↗

PubMed1995

Genistein resistance in human leukaemic CCRF-CEM cells: selection of a diploid cell line with reduced DNA topoisomerase II beta isoform.

Genistein, an isoflavonoid derivative initially described as an in vitro protein tyrosine kinase inhibitor, also inhibits mammalian DNA topoisomerase II both in vitro and in vivo. From a human leukaemic T cell line (CCRF-CEM), two genistein-resistant cell lines, which grow in the presence of 50 and 150 microM genistein, respectively, were selected and designated CEM/GN50 and CEM/GN150. Flow cytometry and karyotype analyses revealed that more than 95% of the parental cells were tetraploid whereas both resistant sublines were essentially diploid and were likely derived from the diploid fraction in the initial population. The CEM/GN cells were 3- to 4-fold resistant to genistein, and highly cross-resistant to certain metabolic inhibitors such as cytosine-arabinoside (50-fold) and 5-fluoro-2'-deoxyuridine (5000-fold). This resistance was associated with a markedly decreased uptake of thymidine and a 10-fold reduction in thymidine kinase activity. The CEM/GM cells were also 15- to 30-fold cross-resistant to topoisomerase inhibitors (etoposide, m-AMSA, 2-Me-9-OH-ellipticinium). Comparison of topoisomerase II activities in the sensitive and resistant cells showed: (i) an approximately 2-fold reduced decatenation activity in nuclear extracts from the resistant cells; (ii) an approximate 30% reduction in DNA-protein cross-links in etoposide-treated resistant cells; and (iii) a markedly reduced expression of the topoisomerase II beta isoform. These data, consistent with our previous results, indicate that the cytotoxicity of genistein is at least in part related to its capacity to inhibit DNA topoisomerase II.

Animals↗

PubMed1995

Morphologic and molecular cytogenetics in neuroblastoma.

BACKGROUND: Some genetic alterations have been shown to have prognostic implication for patients with neuroblastoma: MYCN oncogene amplification, deletion of the short arm of chromosome 1 and di- or tetraploidy. The goal of this study was to analyze these factors in children with neuroblastoma. METHODS: Twenty neuroblastoma samples were analyzed with morphologic cytogenetics, and each of them was compared with MYCN amplification status by Southern blot and fluorescent in situ hybridization (FISH) with a genomic probe. RESULTS: A complete karyotype was obtained for 14 children. A diploid or tetraploid mode and a 1p deletion were found in most children with advanced stages. MYCN amplification status was totally concordant with both methods in all patients, even in a case with low level amplification. A wide intercellular variation in the amplification level in each MYCN amplified sample was shown. CONCLUSION: The use of FISH to assess MYCN amplification rapidly in neuroblastoma is recommended. This method could be very useful in future therapeutic protocols in which treatment is based on MYCN status (and especially for infants and children with localized tumor).

Adolescent↗

PubMed1995

A Tsp509I variant in exon 13 of the neurofibromatosis type 1 (NF1) gene allows the identification of both alleles at the mRNA level.

We describe here a new sequence variant occurring in the coding region of the neurofibromatosis (NF1) gene (exon 13). This exonic polymorphism can be directly investigated by simple restriction enzyme digestion of RT-PCR (reverse transcription-polymerase chain reaction) products, making it a powerful tool for examining allele-specific mRNA expression levels.

Alleles↗

PubMed1995

Evolution of chromosomal alterations and biologic features in two small cell lung carcinoma cell lines established from one patient during the course of the disease.

Two small cell lung cancer (SCLC) cell lines were established from metastases of a patient during the course of the disease. SCLC 74A was derived from biopsy material obtained at the time of diagnosis and SCLC 74B was from a biopsy specimen of a relapsed tumor obtained after treatment. A transition occurred from SCLC 74A, an intermediate form with 5% large cells to SCLC 74B, a standard mixed form with 20% of large cells, with a decrease in neuroendocrine markers and a substantial increase in P-glycoprotein, a multidrug resistance marker. For both cell lines, R-banding and FISH indicated a del(1)(p35pter) also found in other neural-crest-derived tumors, the loss of regions with suspected tumor suppressor genes at 3p, 5q, and 17p, and a recurrent translocation of the 6q24-6qter region to 10p14. Further genetic modifications in SCLC 74B affected chromosomes 2, 3, 5, 10, 11, 14, and 15. The main observations were a der(2)t(2;5)(p16;q?); a der(3;11)(q10;p10) in SCLC 74A which became der(3;14)(q10;p10) and der(11;14)(p10;q10) in SCLC 74B; and the insertion of the 5q13-5q31 region in the der(10)t(6;10). The finding of the same structural abnormalities in both cell lines suggests a monoclonal origin for both metastases. Hypotetraploid cells were in the same proportion as large cells whose number was a characteristic feature of each cell line. They possessed twice the same chromosomal alterations observed in the hypodiploid cells. This suggests a permanent process of tetraploidization.

Aged↗

PubMed1995

Cytogenetic study of a case of synchronous bilateral seminoma.

Cytogenetic analysis of a case of synchronous bilateral seminoma revealed, for each tumor, a chromosomal hypotetraploid mode with gain and loss of chromosomes and only one structural rearrangement, del(1p) and i(12p), which were secondary chromosomal events for the left and right tumors, respectively. This study allowed us to discriminate between a sole or independent origin for these two tumors.

Adult↗

PubMed1995

Neurofibromatosis 1 (NF1) mRNAs expressed in the central nervous system are differentially spliced in the 5' part of the gene.

The neurofibromatosis 1 gene seems to play essential roles at several different stages of life. During embryogenesis, it is involved in cardiac development while in the adult, neurofibromin (the corresponding protein) is mainly expressed in the nervous system, and therein, essentially in neurons, non-myelinating Schwann cells and oligodendrocytes. In addition, the NF1 gene is considered a tumor suppressor gene, since mutations have been associated with the occurrence of benign and malignant tumors in neuralcrest-derived tissues. Using reverse transcription-polymerase chain reaction (RT-PCR) analyses with primers located in exons 7 and 13, we have identified evidence of alternative splicing in this region of the NF1 gene. Cloning and sequencing of cDNA allowed the characterization of an isoform bearing an extra 30 bp sequence between exons 9 and 10a, leading to the insertion of 10 amino acids between residues 420 and 421 of neurofibromin. The insertion is conserved in the mouse. Examination of the pattern of expression of this isoform demonstrated a high level of expression in the central nervous system and an absence of expression in all the other normal tissues tested including peripheral nervous tissues derived from the neural crest. Analysis of brain tumors indicated a reduced expression of the alternative exon in medulloblastomas and oligodendrogliomas. The results presented here are consistent with tissue-specific expression of this alternative exon which we propose to call exon 9br.

Amino Acid Sequence↗

PubMed1995

A new hybrid cell line containing only human chromosome 15 selected through fluorescence in situ hybridization and characterized by Alu-PCR amplification of the human DNA.

Human Cot1 DNA directly labeled with dUTP-fluorochromes (FITC, Rhodamin) and used as a probe, allowed rapid detection of one or a few human chromosomes in human-hamster hybrid cells by in situ hybridization (FISH). A hybrid cell line containing only a human acrocentric chromosome was isolated (CH35B2D). The DNA from this hybrid was used for PCR amplification with a single Alu (A33) primer. After agarose gel electrophoresis, a continuum of intense bands of between 400-700 bp was observed with A33-PCR products. No amplified product was visible with hamster DNA. FISH on normal human metaphases of biotinylated Alu-PCR products obtained with Alu A33 and compétition with human Cot1 DNA showed decoration, with high specificity for chromosome 15. It was identified after R banding obtained with PI or DAPI in an antifade adjusted to pH11 with NaOH. Under the applied conditions, the Alu (A33) products are expected to be useful for characterization, by specific decoration of chromosome 15 aberrations in pathological cells. CH35B2D could be employed for functional studies of genes located on chromosome 15.

Animals↗

PubMed1995

FISH diagnosis of t(8;21) in a myelodysplasia secondary to Hodgkin lymphoma.

A t(8;21)(q22;q22) translocation without blood and bone marrow invasion by immature myeloid precursors was suspected in a conventional karyotype and confirmed by fluorescence in situ hybridization (FISH) with chromosome 8 and 21 painting in a patient previously treated for Hodgkin's lymphoma. Six weeks later, the diagnosis was confirmed by the onset of acute myeloid leukemia (AML) M2 with a t(8;21) in the bone marrow.

Chromosomes, Human, Pair 21↗

PubMed1994

In vitro parameters and treatment outcome in head and neck cancers treated with surgery and/or radiation: cell characterization and correlations with local control and overall survival.

PURPOSE: To determine whether in vivo parameters (surviving fraction at 2 Gy, alpha values, and calculated cell growth fraction) were predictive of the treatment outcome. METHODS AND MATERIALS: Biopsies were obtained from patients with a head and neck tumor. In vitro parameters were determined using the CAM plate assay. Cell characterization by cytogenetic analysis was performed on 19 different cell cultures. In 25 additional cell cultures, cell clonogenicity was tested using the Courtenay Mills assay. RESULTS: Biopsies were obtained from 156 patients with a head and neck tumor and the oropharynx was the predominant primary site. In vitro parameters were obtained in 113 cases (72%) (SF2 in 93 cases and calculated cell growth fraction in 103 cases). Cell characterization showed that cells in CAM plates were diploid with no clonal chromosome abnormalities and gave colonies in soft agar with a mean cloning efficiency of 1.610(-3). Only patients treated with surgery and/or radiation (76), were considered eligible for in vitro parameters and treatment outcome correlation studies. The mean follow-up is over 2 years (range 9-47 months). The local control rate was significantly higher (p = 0.04) for patients with alpha values above the cut-off point of 0.07 Gy-1 (69% vs. 38% at 2 years). The local control rate was also significantly higher (p = 0.04) for patients with calculated cell growth fraction values about the cut-off point of 0.06% (70% vs. 48% at 2 years). Moreover for these latter patients the overall survival rate was also significantly higher (p = 0.004) (54% vs. 26% at 2 years). It is worth noting that alpha and calculated cell growth fraction values below the cut-off points identified a small group of patients (about 20%) who were at a significantly high risk of local failure. From a pragmatic point of view, as only radiosensitivity or calculated cell growth fraction values could be obtained in a certain number of experiments due to technical reasons, the treatment outcome of patients who had either alpha and/or calculated cell growth fraction values below the cut-off levels (about 30% of all patients) was analyzed. This group of patients fared significantly worse (p = 0.02) in terms of local control (50% vs. 68% at 2 years) and (p = 0.04) overall survival (36% vs. 50% at 2 years). CONCLUSION: These results suggest that in vitro parameters using the CAM plate assay, might be useful in predicting the treatment outcome of patients with a head and neck tumor treated with surgery and postoperative radiation, or radiation alone. However, they must be considered as preliminary because the cut offs used in the study were chosen for exploratory purposes. Only a multivariate analysis including all clinical and biologic factors will allow us to draw any firm conclusions.

Carcinoma, Squamous Cell↗