Biomedical subjects

T Cremer

Publications and source records attributed to T Cremer.

At least 55 records · Page 3Linked to original sources

PubMed1997

High-resolution comparative hybridization to combed DNA fibers.

Comparative genomic hybridization (CGH) has proven to be a comprehensive new tool to detect genetic imbalances in genomic DNA. However, the resolution of this method carried out on normal human metaphase spreads is limited to low copy number gains and losses of > or = 10 Mb. An improved resolution allowing the detection of copy number representations of single genes would strongly enhance the applicability of CGH as a diagnostic and research tool. This goal may be achieved when metaphase chromosomes are replaced by an array of target DNAs representing the genes of interest. To explore the feasibility of such a development in a model system we used cosmid MA2B3, which encompasses about 35 kb in the vicinity of exon 48 of the human dystrophin gene. Linearized cosmid fibers were attached to a glass surface and aligned in parallel by "molecular combing". Two-color fluorescence in situ suppression hybridization was performed on these cosmid fibers with probe mixtures containing different ratios (ranging from 1:2 to 4:1) of biotin- and digoxigenin-labeled MA2B3 cosmid DNAs. For each mixture fluorescence ratios were determined for 40-50 individual combed DNA molecules. In two series comprising a total of 651 molecules the median fluorescence ratio measurements revealed a linear relationship with the chosen probe ratios. Our study demonstrates that fluorescence ratio measurements on single DNA molecules can be performed successfully.

Biotin↗

PubMed1997

Removal of repetitive sequences from FISH probes using PCR-assisted affinity chromatography.

The vast majority of probes used in fluorescence in situ hybridization (FISH) contain repetitive DNA. This DNA is usually competed out of a hybridization reaction by the addition of an unlabeled blocking agent, Cot-1 DNA. We have successfully removed repetitive DNA from two complex FISH probe sets: a degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) single human chromosome library and genomic DNA. The procedure involved hybridizing in solution a DOP-PCR-amplifiable probe set with a 50-fold excess of biotin-labeled Cot-1 DNA, and capturing the Cot-1 DNA-containing hybrids using streptavidin magnetic particles, followed by purification and reamplification of the unbound fraction. Probes were checked for depletion of repeats by hybridization to chromosomes without Cot-1 DNA. Results showed hybridization patterns comparable to those achieved with untreated probes hybridized with Cot-1 DNA.

Chromatography, Affinity↗

PubMed1997

Comparative genomic hybridization analysis of human neuroblastomas: detection of distal 1p deletions and further molecular genetic characterization of neuroblastoma cell lines.

Deletions of the short arm of chromosome 1 and MYCN amplification are the most frequently encountered genetic changes in disseminated neuroblastomas and neuroblastoma cell lines. Different strategies have been followed for detection of these and other genomic changes in neuroblastoma including karyotyping, FISH, and LOH, each with its own limitations. Here we report upon the evaluation of comparative genomic hybridization (CGH) in the analysis of neuroblastoma cell lines, with the emphasis on the assessment of the reliability of CGH for the detection of distal 1p deletions. We have analyzed seven neuroblastoma cell lines for which the 1p status was previously studied in detail using FISH and LOH. Our results show that CGH allows reliable detection of distal 1p deletions, including a small interstitial deletion in cell line SK-N-AS. Furthermore, CGH also allows the detection of chromosomal imbalance which would otherwise remain undetected, and provides useful information for further molecular characterization of chromosomal imbalances.

Child, Preschool↗

PubMed1997

Matrix-based comparative genomic hybridization: biochips to screen for genomic imbalances.

Comparative genomic hybridization (CGH) to metaphase chromosomes has been widely used for the genome-wide screening of genomic imbalances in tumor cells. Substitution of the chromosome targets by a matrix consisting of an ordered set of defined nucleic acid target sequences would greatly enhance the resolution and simplify the analysis procedure, both of which are prerequisites for a broad application of CGH as a diagnostic tool. However, hybridization of whole genomic human DNA to immobilized single-copy DNA fragments with complexities below the megabase pair level has been hampered by the low probability of specific binding because of the high probe complexity. We developed a protocol that allows CGH to chips consisting of glass slides with immobilized target DNAs arrayed in small spots. High-copy-number amplifications contained in tumor cells were rapidly scored by use of target DNAs as small as a cosmid. Low-copy-number gains and losses were identified reliably by their ratios by use of chromosome-specific DNA libraries or genomic fragments as small as 75 kb cloned in PI or PAC vectors as targets, thus greatly improving the resolution achievable by chromosomal CGH. The ratios obtained for the same chromosomal imbalance by matrix CGH and by chromosomal CGH corresponded very well. The new matrix CGH protocol provides a basis for the development of automated diagnostic procedures with biochips designed to meet clinical needs.

Chromosome Aberrations↗

PubMed1996

Characterization of genomic alterations associated with glioma progression by comparative genomic hybridization.

Genomic alterations associated with glioma progression were determined by comparative genomic hybridization (CGH) 30 tumors from 15 patients with primary gliomas of World Health Organization (WHO) grade II that on recurrence showed progression to malignant gliomas of WHO grades III or IV (five cases of astrocytoma grade II (A II) to grade III (AA III), five cases of A II to glioblastoma multiforme grade IV (GBM) and five cases of oligodendroglioma grade II (O II) to grade III (AO III)). All tumors were additionally screened for p53 mutations by single strand conformational polymorphism and heteroduplex analysis of exons 5-8, followed by direct sequencing. Mutations of p53 were found in the primary and recurrent tumors of all cases of A II progressing to GBM and three of five cases of A II recurring as AA III. Alterations identified by CGH in more than one primary A II included losses on Xp (3/10) and 5p (2/10), gains on 8q and 19p (2/10 each), and gain/amplification on 12p (2/10). Common progression associated changes found in AA III or GBM were losses on 4q, 9p, 10q, 11p, 13q (4/10 each) and gains on 1q, 6p, 20q (2/10 each). The most frequent amplification site was located on 12p13 (1/10 A II, 3/5 AA III, 1/5 GBM). Other amplified chromosomal regions were 13q32-q34 (1/10 AII, 2/5 GBM), 7q31-qter (1/5 AA III, 1/5 GBM), 12q22-qter and 18p (1/5 AA III). In contrast to the astrocytic gliomas, only one of five oligodendroglial cases showed a p53 mutation. Genetic abnormalities identified by CGH to occur more than once were restricted to four chromosomes (1, 4, 9 and 19). Our results provide a comprehensive overview of the genomic alterations associated with the progression of individual gliomas and substantiate the hypothesis that glioma progression is associated with a cumulative acquisition of multiple genetic changes.

Adult↗

PubMed1996

Recurrent gain of chromosome arm 7q in low-grade astrocytic tumors studied by comparative genomic hybridization.

Consistent tumor-specific chromosomal aberrations have not been described in low-grade astrocytic tumors. The most frequent genetic alterations are mutations of the TP53 tumor suppressor gene and/or loss of heterozygosity (LOH) on 17p that occur in about 30% of the cases in adult patients but that are uncommon in childhood tumors. We used comparative genomic hybridization (CGH) to map DNA copy number alterations in 18 primary low-grade astrocytic tumors (ten adult patients and eight children). A gain of chromosome arm 7q was the most frequent event detected in five of ten astrocytomas (50%) from adult patients, followed by DNA amplification on chromosome arm 8q and gain on 12p (two cases). Loss of chromosomal regions on 1p, 4q, and the X chromosome was observed in two of ten cases [including one patient afflicted with Turner syndrome (45,X)]. In contrast, no consistent changes were observed in low-grade astrocytomas in children. A loss of the X chromosome was the sole aberration detected in two of eight cases using DNA extracted from the normal brain tissue. The findings suggest that a gain of 7q is an early event in the initiation of astrocytomas in adult patients. The discrepant findings in low-grade astrocytic tumors in adults compared to tumors in children support the the hypothesis that there might be different mechanisms responsible for tumor development.

Adolescent↗

PubMed1996

Characterization of double minute chromosomes' DNA content in a human high grade astrocytoma cell line by using comparative genomic hybridization and fluorescence in situ hybridization.

The presence of double minute chromosomes (dmin) in cancer cells is known to be correlated with gene amplifications. In human high grade astrocytomas or glioblastomas, about 50% of cytogenetically characterized cases display dmin. G5 is a cell line which has been established from a human glioblastoma containing multiple dmin. In order to identify the DNA content of these dmin, three techniques were successively used: conventional cytogenetic analysis, comparative genomic hybridization (CGH). and fluorescent in situ hybridization (FISH). The karyotype of G5 cells showed numerical chromosome changes (hypertriploidy), several marker chromosomes, and multiple dmin. CGH experiments detected two strong DNA amplification areas located in 9p21-22 and 9p24, as well as an underrepresentation of chromosomes 6, 10, 11, 13, 14, and 18q. By using FISH with a chromosome 9-specific painting probe to metaphase chromosomes of the G5 cell line, dmin were shown to contain DNA sequences originating from chromosome 9. This study demonstrates the usefulness of a combination of classical karyotyping, CGH, and FISH to identify the chromosomal origin of amplified DNA sequences in dmin.

Astrocytoma↗

PubMed1996

Nuclear architecture and the induction of chromosomal aberrations.

Progress in fluorescence in situ hybridization, three dimensional microscopy and image analysis has provided the means to study the three-dimensional structure and distribution of chromosome territories within the cell nucleus. In this contribution, we summarize the present state of knowledge of the territorial organization of interphase chromosomes and their topological relationships with other macromolecular domains in the human cell nucleus, and present data from computer simulations of chromosome territory distributions. On this basis, we discuss models of chromosome territory and nuclear architecture and topological consequences for the formation of chromosome exchanges.

Animals↗

PubMed1996

Chromosome aberration analysis in atomic bomb survivors and Thorotrast patients using two- and three-colour chromosome painting of chromosomal subsets.

Chromosomal translocations in peripheral lymphocytes of three healthy Hiroshima atomic (A)-bomb survivors, as well as three Thorotrast patients and two non-irradiated age-matched control persons from the German Thorotrast study were studied by two- and three-colour fluorescence in situ hybridization (chromosome painting) with various combinations of whole chromosome composite probes, including chromosomes 1, 2, 3, 4, 6, 7, 8, 9 and 12. Translocation frequencies detected by chromosome painting in cells of the A-bomb survivors were compared with results obtained by G-banding. A direct comparison was made, i.e. only those cells with simple translocations or complex aberrations detected by G-banding were taken into consideration which in principle could be detected also with the respective painting combination. The statistical analysis revealed no significant differences from a 1:1 relationship between the frequencies of aberrant cells obtained by both methods. The use of genomic translocation frequencies estimated from subsets of chromosomes for biological dosimetry is discussed in the light of evidence that chromosomes occupy distinct territories and are variably arranged in human lymphocyte nuclei. This territorial organization of interphase chromosomes implies that translocations will be restricted to chromatin located at the periphery of adjacent chromosome territories.

Aged↗

PubMed1996

Centromere and telomere movements during early meiotic prophase of mouse and man are associated with the onset of chromosome pairing.

The preconditions and early steps of meiotic chromosome pairing were studied by fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes to mouse and human testis tissue sections. Premeiotic pairing of homologous chromosomes was not detected in spermatogonia of the two species. FISH with centromere- and telomere-specific DNA probes in combination with immunostaining (IS) of synaptonemal complex (SC) proteins to testis sections of prepuberal mice at days 4-12 post partum was performed to study sequentially the meiotic pairing process. Movements of centromeres and then telomeres to the nuclear envelope, and of telomeres along the nuclear envelope leading to the formation of a chromosomal bouquet were detected during mouse prophase. At the bouquet stage, pairing of a mouse chromosome-8-specific probe was observed. SC-IS and simultaneous telomere FISH revealed that axial element proteins appear as large aggregates in mouse meiocytes when telomeres are attached to the nuclear envelope. Axial element formation initiates during tight telomere clustering and transverse filament-IS indicated the initiation of synapsis during this stage. Comparison of telomere and centromere distribution patterns of mouse and human meiocytes revealed movements of centromeres and then telomeres to the nuclear envelope and subsequent bouquet formation as conserved motifs of the pairing process. Chromosome painting in human spermatogonia revealed compacted, largely mutually exclusive chromosome territories. The territories developed into long, thin threads at the onset of meiotic prophase. Based on these results a unified model of the pairing process is proposed.

Animals↗

PubMed1996

Active and inactive genes localize preferentially in the periphery of chromosome territories.

The intranuclear position of a set of genes was analyzed with respect to the territories occupied by the whole chromosomes in which these genes are localized. Genes and their respective chromosome territories were simultaneously visualized in three-dimensionally preserved nuclei applying dual color fluorescence in situ hybridization. Three coding (DMD, MYH7, and HBB) and two noncoding sequences (D1Z2 and an anonymous sequence) were analyzed in four different cell types, including cells where DMD and MYH7 are actively transcribed. Spatial analysis by confocal laser scanning microscopy revealed that the genes are preferentially located in the periphery of chromosome territories. This positioning was independent from the activity of the genes. In contrast, the non-expressed anonymous fragment was found randomly distributed or localized preferentially in the interior of the corresponding chromosome territory. Furthermore, the distribution of the analyzed genes within the territorial peripheries was found to be highly characteristic for each gene, and, again, independent from its expression. The impact of these findings with regard to the three-dimensional arrangement of the linear DNA string within chromosome territories, as well as with respect to a putative nuclear subcompartment confining gene expression, are discussed.

Cells, Cultured↗

PubMed1996

Three-dimensional reconstruction of painted human interphase chromosomes: active and inactive X chromosome territories have similar volumes but differ in shape and surface structure.

This study provides a three-dimensional (3D) analysis of differences between the 3D morphology of active and inactive human X interphase chromosomes (Xa and Xi territories). Chromosome territories were painted in formaldehyde-fixed, three-dimensionally intact human diploid female amniotic fluid cell nuclei (46, XX) with X-specific whole chromosome compositive probes. The colocalization of a 4,6-diamidino-2-phenylindole dihydrochloride-stained Barr body with one of the two painted X territories allowed the unequivocal discrimination of the inactive X from its active counterpart. Light optical serial sections were obtained with a confocal laser scanning microscope. 3D-reconstructed Xa territories revealed a flatter shape and exhibited a larger and more irregular surface when compared to the apparently smoother surface and rounder shape of Xi territories. The relationship between territory surface and volume was quantified by the determination of a dimensionless roundness factor (RF). RF and surface area measurements showed a highly significant difference between Xa and Xi territories (P < 0.001) in contrast to volume differences (P > 0.1). For comparison with an autosome of similar DNA content, chromosome 7 territories were additionally painted. The 3D morphology of the chromosome 7 territories was similar to the Xa territory but differed strongly from the Xi territory with respect to RF and surface area (P < 0.001).

Amniotic Fluid↗

PubMed1996

Clinically distinct subgroups of glioblastoma multiforme studied by comparative genomic hybridization.

Studies investigating genetic alterations potentially constituting prognostic factors in glioblastoma multiforme (GBM) have centered mainly around amplification events. Comparative genomic hybridization (CGH) is a recent molecular cytogenetic technique that allows the detection of chromosomal imbalances and amplification sites in tumor DNA prepared from fresh or archival material. A group of 94 patients with GBM underwent surgery followed by a standard course of radiotherapy. Neuroradiologic monitoring with gadolinium-enhanced serial magnetic resonance imaging was applied to study the radiologically progression-free interval (RPFI) and tumor regrowth velocity. These parameters provided a clinical estimate of the postoperative tumor regrowth kinetics and yielded two clinically distinct groups. The most pronounced cases were selected from each group, i.e., those with the most favorable and unfavorable prognosis. Two subgroups with a statistically significant difference in RPFI (p < 0.001, Mann-Whitney U test) containing 10 patients each were formed: Subgroup A (slow tumor regrowth kinetics) and subgroup B (fast tumor regrowth kinetics). For a search of chromosomal alterations that might be correlated with tumor regrowth kinetics, we applied CGH to formalin-fixed, paraffin-embedded tumor tissue from these 20 patients. Except for autosomes 18 and 21, all chromosomes were involved at least once in copy-number aberrations. Events commonly associated with GBM, i.e., gains of chromosome 7, complete and partial losses of 9p, 10, and 22q, were not distributed differently between the two subgroups. The following differences were noticeable. Gains (including amplifications) of 12q14-q21 and of 19 were observed more often in subgroup A. Losses of 6q16-qter and parts of 13, and gains of 20, were more frequent in subgroup B. RPFI was significantly shorter for patients without amplification sites than for patients with gene amplification. RPFI did not differ significantly between patients with or without 7p12 amplification, where the epidermal growth factor receptor gene is localized. New amplification sites for GEM tumors were revealed at 11q13 and 11q22-q23. Loss of chromosome 10 was restricted to bands 10q25-q26 in one case. Although differences in the copy-number karyotypes of patients with slow and fast postoperative tumor-regrowth kinetics were noted, the present CGH study did not reveal any single alteration useful as a prognostic factor. In particular, these data do not support the assumption that patients suffering from GBM with amplification events would have a poorer prognosis than others.

Adult↗

PubMed1995

Comparative genomic hybridization of formalin-fixed, paraffin-embedded breast tumors reveals different patterns of chromosomal gains and losses in fibroadenomas and diploid and aneuploid carcinomas.

Comparative genomic hybridization serves as a screening test for regions of copy number changes in tumor genomes. We have applied the technique to map DNA gains and losses in 33 cases of formalin-fixed, paraffin-embedded primary breast tumors (13 fibroadenomas and 10 diploid and 10 aneuploid carcinomas). No genomic imbalances were found in fibroadenomas. Recurrent findings in adenocarcinomas include copy number increases for chromosomes 1q (14 of 20 samples), 8q (10 of 20), 17q (5 of 20), 6p (3 of 20), 13q (3 of 20), and 16p (3 of 20), and copy number decreases for chromosomes 22 (7 of 20), 17p (6 of 20), and 20 (3 of 20). Regional high level copy number increases were observed on chromosome bands 1q32, 8p11, 8q24, 10p, 11q13, 12p, 12q15, 17q11-12, and 17q22-24. The majority of the samples were studied for gene amplification of c-myc, c-erbB2, cycD1, and int-2 by means of Southern blot analysis. The comparison with DNA ploidy measurements revealed a different distribution and a significantly higher number of chromosomal aberrations in aneuploid tumors than in diploid tumors and in fibroadenomas.

Aneuploidy↗

PubMed1995

Quantitative analysis of comparative genomic hybridization.

Comparative genomic hybridization (CGH) is a new molecular cytogenetic method for the detection of chromosomal imbalances. Following cohybridization of DNA prepared from a sample to be studied and control DNA to normal metaphase spreads, probes are detected via different fluorochromes. The ratio of the test and control fluorescence intensities along a chromosome reflects the relative copy number of segments of a chromosome in the test genome. Quantitative evaluation of CGH experiments is required for the determination of low copy changes, e.g., monosomy or trisomy, and for the definition of the breakpoints involved in unbalanced rearrangements. In this study, a program for quantitation of CGH preparations is presented. This program is based on the extraction of the fluorescence ratio profile along each chromosome, followed by averaging of individual profiles from several meta phase spreads. Objective parameters critical for quantitative evaluations were tested, and the criteria for selection of suitable CGH preparations are described. The granularity of the chromosome painting and the regional inhomogeneity of fluorescence intensities in metaphase spreads proved to be crucial parameters. The coefficient of variation of the ratio value for chromosomes in balanced state (CVBS) provides a general quality criterion for CGH experiments. Different cutoff levels (thresholds) of average fluorescence ratio values were compared for their specificity and sensitivity with regard to the detection of chromosomal imbalances.

Chromosome Banding↗

PubMed1995

Correlation of microscopic phenotype with genotype in a formalin-fixed, paraffin-embedded testicular germ cell tumor with universal DNA amplification, comparative genomic hybridization, and interphase cytogenetics.

We present a strategy for the evaluation of numerical copy number changes of DNA segments within a solid tumor genome that allows the correlation of microscopic phenotype with genotype in formalin-fixed, paraffin-embedded tumor material. Cells from a human testicular germ cell tumor and adjacent tissue areas with normal seminiferous tubules were selected separately from microscopically analyzed histological tissue sections, and DNA was extracted from the selected areas. After universal DNA amplification, the amplification products were subjected to comparative genomic hybridization. The results confirmed balanced chromosome copy numbers for the normal tissue area, although the analysis of the tumor tissue area revealed numerous gains and losses of chromosome segments. The comparative genomic hybridization results were used to select DNA probes for interphase cytogenetics on serial sections. We conclude that this technique allows the screening of selected tissue areas for numerical DNA alterations, thus enabling a direct phenotype-genotype comparison.

Adult↗

PubMed1994

Chromosomal gains and losses in uveal melanomas detected by comparative genomic hybridization.

Eleven uveal melanomas were analyzed using comparative genomic hybridization (CGH). The most abundant genetic changes were loss of chromosome 3, overrepresentation of 6p, loss of 6q, and multiplication of 8q. The smallest overrepresented regions on 6p and 8q were 6pter-->p21 and 8q24-->qter, respectively. Several additional gains and losses of chromosome segments were repeatedly observed, the most frequent one being loss of 9p (three cases). Monosomy 3 appeared to be a marker for ciliary body involvement. CGH data were compared with the results of chromosome banding. Some alterations, e.g., gains of 6p and losses of 6q, were observed with higher frequencies after CGH, while others, e.g., 9p deletions, were detected only by CGH. The data suggest some similarities of cytogenetic alterations between cutaneous and uveal melanoma. In particular, the 9p deletions are of interest due to recent reports about the location of a putative tumor-suppressor gene for cutaneous malignant melanoma in this region.

Adult↗

PubMed1994

Mapping of multiple DNA gains and losses in primary small cell lung carcinomas by comparative genomic hybridization.

Comparative genomic hybridization was applied for a comprehensive screening of under- and overrepresentation of genetic material in 13 autoptic small cell lung cancer specimens. The most abundant genetic changes include DNA losses of chromosome arms 3p, 5q, 10q, 13q, and 17p and DNA gains of 3q, 5p, 8q, and 17q. Amplification sites in these tumors were mapped to 22 chromosome bands. The most frequently involved band was 19q13.1 (4 cases). Bands 1p32, 2p23, 7q11.2, 8q24, and 13q33-34 were involved in two cases each.

Autopsy↗