Biomedical subjects

U Claussen

Publications and source records attributed to U Claussen.

At least 73 records · Page 4Linked to original sources

PubMed1999

P53 genotyping - an effective concept for molecular testing of head and neck cancer?

P53 mutations are currently recognized as the most common genetic alteration in human tumors. The purpose of our study was to evaluate the significance and reliability of p53 genotyping in head and neck cancer as a possible marker permitting the prediction of tumor behavior and clinical outcome. P53 genotyping in our study refers to highly sensitive molecular screening in order to detect structural alterations in the nucleic acid sequence of the gene. Exons 2-11 and adjacent intronic regions were screened for mutations by direct genomic sequencing or by bi-directional dideoxyfingerprinting in 66 primary tumors of the larynx, pharynx and oral cavity. Alterations in the of the p53 gene were detected in 36% (24 of 66) of the analyzed tumors, no mutation was found in our cohort outside exons 5-8. The frequency of p53 mutation had no correlation to the tumor stage or tumor site. The recurrence rate in patients with a p53 alteration was not significantly higher compared to patients without a p53 mutation in their primary tumors. Summarizing the results of our study only limited reliability of p53 genotyping as an effective concept for molecular testing of head and neck cancer was found.

Biomarkers, Tumor↗

PubMed1999

Interphase cytogenetic diagnosis of bladder cancer on cells from urine and bladder washing.

In order to determine the value of fluorescence in situ hybridization (FISH) in the diagnosis and follow-up of bladder cancer interphase cytogenetics was performed on cells from urine and bladder washings. 50 ml of urine or bladder washings were collected. FISH was carried out using centromere probes for chromosomes 7, 8, 9 and 12 according to standard protocols. In each case 100 cell nuclei were analysed. Fifty-four samples from urine and 67 samples from bladder washing were analysed by FISH in comparison with results obtained by conventional cytology. Sensitivity of detection of tumor cells by FISH was 68.5% in urine and 63% in bladder washings regardless of tumor stage and grade. Sensitivity obtained by conventional cytology was 50% in urine and 77.3% in bladder washings. FISH on cells from urine samples is an effective complement to the standard urine cytology. Using centromere probes this approach is characterized by high specificity and sensitivity in tumors with T-category higher than pTa and grade higher than G1.

Carcinoma, Transitional Cell↗

PubMed1999

Determination of telomerase activity in multifocal renal cell carcinoma.

In a consecutive series of 245 patients with renal cell carcinoma (RCC) all nephrectomy specimens were examined regarding the presence of additional multifocal lesions. Therefore, 3-mm step sections were performed and investigated by macroscopical and histopathological examination. Twenty-six multifocal tumors were found in 19 specimens by this procedure. In order to characterize the biological activity of these multifocal lesions in more detail, their telomerase activity was analyzed. Fourteen of the 23 investigated multifocal tumors (60.9%) displayed telomerase activity. In three cases the tissue probes were too small for further investigations. Telomerase activity was also detected in 10 out of 15 (66.7%) matched primary renal cell carcinomas. In contrast, all corresponding normal kidney cortex tissues used as control were telomerase negative. From our results we conclude, that small tumor lesions of RCC must be expected to have a malignant potential similar to that of primary carcinomas. Hence, nephron-sparing surgery cannot be routinely carried out even in patients with small tumors.

Carcinoma, Renal Cell↗

PubMed1999

Molecular cytogenetic characterization of a prenatally detected supernumerary minute marker chromosome 8.

The characterization of a prenatally detected very small (approximately half of 18p-(karyotype: 47,XX,+mar[16]/46,XX[7]) supernumerary marker chromosome (SMC) identified by GTG-banding analysis is described. The marker has been identified as derived from chromosome 8 centromeric material using a combination of different cytogenetic (GTG-, NOR-, CBG banding), molecular cytogenetic (24 colour-fluorescent in situ hybridization [FISH], three-colour FISH using centromeric probes for all human chromosomes) and molecular genetic techniques (microsatellite analysis). This is the first case described with such a minute SMC derived from chromosome 8 diagnosed prenatally, the 15th case reporting on a SMC originating from chromosome 8 and the third such case without any severe clinical features.

Adult↗

PubMed1997

Cytogenetic, histopathologic, and immunologic studies of multifocal renal cell carcinoma.

BACKGROUND: Multifocal tumor areas occurred in 12-22% of patients with renal cell carcinoma. It is unknown whether these tumors have malignant potential and characterize a higher risk for metastases. The performance of nephron-sparing surgery in patients with low grade or low stage tumors is controversial. METHODS: Primary and secondary tumors were analyzed by conventional cytogenetics and fluorescence in situ hybridization. Production of interleukin (IL)-6, IL-10, IL-11, and transforming growth factor (TGF)-beta1 were determined using standard enzyme-linked immunosorbent assay and bioassays. RESULTS: In 15.2% of the renal cell carcinoma cases evaluated, multifocal tumors were detected. Cytogenetics revealed a concordance of primary and secondary tumors in 9 of 14 cases (64%). In 11 of 12 multifocal tumors (94%), the same immunologic activity status was observed in both primary and secondary tumors. CONCLUSIONS: Secondary tumors must be expected to have malignant potential similar to that of the primary tumors. This was underscored by the high concordance of cytogenetic, histopathologic, and immunologic data in this study.

Carcinoma, Renal Cell↗

PubMed1997

Determination of the origin of single nucleated cells in maternal circulation by means of random PCR and a set of length polymorphisms.

Non-invasive prenatal diagnosis on fetal nucleated erythrocytes from the maternal circulation is hampered by the small number of nucleated erythrocytes and the uncertainty as to whether they are of fetal or maternal origin. To overcome the latter limitation, single nucleated erythrocytes were separated and enriched from maternal blood by a triple density gradient and a monoclonal antibody (CD71) in combination with a magnetic activated cell sorter. Single nucleated cells were microscopically examined, individually collected with extended Pasteur pipettes, and each transferred into separate caps for the polymerase chain reaction (PCR). The DNA of the single nucleated erythrocytes was amplified at least 50-fold with a random PCR technique, viz., primer extension preamplification. Precise differentiation between maternal and fetal nucleated erythrocytes was achieved via PCR by using primers flanking highly polymorphic nucleotide repeats (DIS53, ACTBP2 and D21S11) and with a XY-specific primer pair (amelogenin). A total of 134 putative nucleated erythrocytes were analyzed from blood samples of 19 pregnant women. With the help of the polymorphic repeats, 25% were assigned as being of maternal origin, 26% of fetal origin, and 48% were uninformative. In cases with male fetuses, the amelogenin primers revealed 30% of cells to be fetal nucleated erythrocytes, the remaining 70% being of maternal origin. The results indicate that the combination of random PCR and PCR-mediated polymorphism analysis on the DNA of single nucleated erythrocytes is a useful technique for non-invasive prenatal diagnosis.

Amelogenin↗

PubMed1997

Isolation of DNA from the centromere of human chromosome 7 by microdissection.

Centromeres remain the least characterized regions of human chromosomes because they have a very high content of repetitive DNA. Here, we describe a microdissection library from the centromeric region of human chromosome 7 and its use for generating sequence tagged sites (STSs). The library contains about 1500 clones with an average insert size of 150 bp and only about 15% of the clones harbour repetitive human DNA. Seven clones hybridizing to alphoid DNA were found to correspond to a fragment of the D7Z2 alphoid array on chromosome 7, thus confirming the origin of the library. A number of clones not containing known repetitive DNA were used to generate STSs that identified yeast artificial chromosomes (YACs) and in turn allowed the STSs to be placed on the physical map. One STS is located between the two Genethon genetic markers closest to the centromere on the q side. Another STS was located 3-4cM away in 7q11.2, while a third identified YACs containing both low-copy and alphoid sequences that are not yet mapped but are clearly centromeric. The library therefore comprises a collection of sequences from the centromeric region of chromosome 7 that can be used to generate STSs and to map the entire centromeric region.

Amniotic Fluid↗

PubMed1997

The spreading of metaphases is a slow process which leads to a stretching of chromosomes.

In routine chromosome harvesting of blood lymphocytes it is well accepted that metaphase spreads are obtained from fixed mitotic cells which burst on the surface of slides during the dropping procedure. For confirmation and clarification, fixed mitotic cells were dropped onto coverslips and observed under an inverted microscope during the evaporation of the fixative. Fixed mitotic cells in the metaphase stage first stick onto the surface of the coverslip without changing their three-dimensional shape and they do not burst. Thereafter, when evaporation of the fixative occurs, they slowly flatten until they are spread. This slow process leads to a stretching of chromosomes which may be a prerequisite for high resolution banding patterns. Confocal laser scanning microscopic measurements of the length, thickness, and width of chromosomes after (i) short term evaporation of the fixative, (ii) evaporation of the fixative under routine harvesting conditions and (iii) a prolonged evaporation, confirmed the stretching of chromosomes. The humidity, the temperature, and the drying time of the fixative influence the dynamic flow of the remaining fixative on the slide. This dynamic flow leads to an intensive wash of the fixed mitotic cells with increasing concentrations of acetic acid which is primarily responsible for the better quality of the metaphase spread.

Cell Nucleus↗

PubMed1997

The nature of G-bands analyzed by chromosome stretching.

To investigate the nature of G-banding, chromosome stretching was performed on chromosome 6 at the 400-band level of normal human lymphocytes that had been cultivated and harvested using standard techniques. The GTG-banding patterns of five stretched chromosomes 6 were compared microscopically with each other and found to be identical at the 1,400-band level. A high-resolution ideogram at the 1,400-band level was constructed. The banding pattern at this level appeared to be very regular, with all dark bands at the 400-band level splitting into three to six dark subbands. While the dark subbands observed at the 1,400-band level seem to derive solely from the dark bands seen at the 400-band level, light bands visible at the 400-band level do not split into subbands, which is in contrast to the published (ISCN, 1995) ideograms. The splitting process, which was analyzed on the video monitor in more detail, shows that chromosome stretching is due mainly to the appearance of light subbands flanked by dark subbands. To shed more light on this phenomenon, the staining intensity of the dark bands and their subbands was measured while the chromosomes were stretched from the 400- to the 1,400-band level. At first, staining intensity was found to diminish in inverse proportion to the elongation of the chromosome, but then remained relatively unaffected until the dark subbands were gradually split up. After stretching to the 1,400-band level, these dark subbands were followed by newly appearing small light subbands, which were about the same size as the stretched light bands visible at the 400-band level. The results indicate that, in general, the light bands of human chromosomes are the preferentially stretched chromosome regions and that the resolution-dependent characteristic banding pattern of human chromosomes is mainly based on a fixed hierarchy of the stretchability of chromosomes.

Centromere↗

PubMed1997

Prenatal diagnosis of a half-cryptic translocation using chromosome microdissection.

This report describes a case of a paternal balanced, but apparently non-reciprocal, insertion of chromosome 15 material into the short arm of chromosome 17 with difficulties in distinguishing between the normal and the deleted chromosome 15 in prenatal karyotype analysis. Microdissection and degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) of the paternal 17p+ chromosome was performed to generate a painting probe specific for the small region inserted from chromosome 15 into chromosome 17. Fluorescence in situ hybridization (FISH) of this probe simultaneously with a differentially labelled 15q microdissection probe enabled the identification of a balanced karyotype in the fetus. In this case, microdissection combined with FISH was the only method for obtaining a reliable result within the short time available for prenatal diagnosis. In addition, it was possible to identify with certainty the originally suspected reciprocal translocation as an insertion of the region 15q22.3-->q23 or 24 into the sub-telomeric region of 17p [ins(17;15)(p13;q22.3q23 or 24)]. Thus, the chromosomal defect of two family members with a partial trisomy of chromosome 15 having severe mental retardation and dysmorphic features was identified precisely.

Adult↗

PubMed1996

Interstitial deletion of chromosome 6q: precise definition of the breakpoints by microdissection, DNA amplification, and reverse painting.

Routine chromosomal analysis using GTG-banding alone showed a mosaic terminal deletion of 6q in a 14-week-old boy with developmental retardation, facial anomalies, agenesis of corpus callosum, cleft palate, hypotonia, short neck and pterygium colli, and minor anomalies of hands and feet. Discrepancies between the clinical findings on our patient and those described in the literature on patients having terminal deletions led to a more precise analysis of the karyotype. Reverse painting was performed on normal G-banded metaphases for exact determination of the breakpoints and on metaphases of the patient for evaluation of mosaicism. A DNA library that was obtained by microdissection of three deleted chromosomes 6 was used as a painting probe. Subsequent DNA amplification was performed with the help of topoisomerase-pretreated degenerate oligonucleotide primers. Unexpectedly, the hybridization pattern on normal metaphase chromosomes revealed an interstitial deletion with breakpoints at 6q25.1 and 6q27 instead of a terminal deletion. Hybridization on metaphases of the patient showed one deleted chromosome 6 in all metaphases analyzed at a higher resolution rather than mosaicism as previously assumed [karyotype, 46,XY,del(6)(q25.1 --> q27)]. We assume that in the single cases of 6q- described in the literature the deletions are misclassified. This might be due to difficulties in distinguishing between interstitial and terminal deletions at 6q and in precisely defining chromosomal breakpoints after GTG-banding alone.

Abnormalities, Multiple↗

PubMed1996

Changes in telomere lengths in renal cell carcinomas.

Telomeres, the extreme ends of chromosomes, play an important role in chromosome structure and function. The shortening of telomeres is one of the supposed mechanisms of cellular aging and death. Because of end replication problems the length of telomeres decreases with every cell cycle. This may lead to chromosome instability and additional genetic alterations possibly responsible of significant tumor development. In many cancer cells the length of telomeres depends on a balance between the loss of telomeric repeats, at each replication cycle, and the telomere lengthening, by the enzyme telomerase, which is repressed in most normal somatic cells. Many tumor cells demonstrate shortened telomeres in comparison to the corresponding normal tissue. In some types of human cancers the reduction of telomeric repeats was correlated with increasing disease severity. We analyzed Southern blots of HINF1-digested DNA of a large number of renal cell carcinomas (RCC) including different tumor areas, secondary tumors and metastases (76 cases with 142 tumor samples) for changes in the length of telomeric repeats using the oligonucleotide probe (TTAGGG)3 and found telomere shortening in 54%, suggesting that a reduction of the telomeric repeat length is not a general characteristic in RCC. Intratumor heterogeneity was demonstrated in seven cases. But also two RCC, with elongated telomeres in the tumor tissue, were observed. Shortened telomeres do not seem to be associated with advanced stages of tumor development or specific histopathological subtypes of RCC.

Adult↗

PubMed1996

[Cyto- and molecular genetic studies of renal cell carcinoma with special reference to multifocal carcinoma lesions].

In 86 consecutive radical nephrectomies we found in 12 kidneys (13.8%) a total of 17 multicentric foci of carcinoma. Beside the usual histopathological investigations we performed in seven secondary tumors further cyto-and molecular genetic analyses. The structural and/or numerical chromosome aberrations in the small secondary tumors were similar to the changes in the main tumors. We conclude that these foci have malignant potential. In eight of these results and the multicentricity that was found, we continue to recommend elective radical nephrectomy in renal cell carcinoma.

Aged↗

PubMed1995

[Rapid karyotyping in the 2nd and 3rd trimester: results and experiences].

Rapid karyotyping in the second and third trimester is an increasing field of collaboration between women's hospitals and human genetics. Techniques available for rapid karyotyping are: 1. Amniocentesis; to obtain amniotic fluid cells for culturing and subsequent chromosome harvesting using the pipette method or the "in situ" technique. The average time between preparation of the amniotic fluid and the verbal notification of the analysed karyotype is 4.65 days for the pipette method and 5.97 days for the "in situ" technique. The major advantages are that amniocentesis can be handled safely by many gynaecologist, and the amniotic fluid samples can be posted easily to cytogenetic units familiar with rapid karyotyping. The main disadvantage is that currently only a few laboratories are able to handle the pipette method or the "in situ" technique for rapid karyotyping. 2. Fetal blood sampling (cordocentesis); and subsequent chromosome analysis on cultivated fetal lymphocytes leading to results within 2 to 4 days. The main advantage of this procedure is the reliability of the results obtained. Fetal blood sampling, however, is restricted to specialists; this may involve scheduling delays. 3. Placental biopsy; with subsequent direct preparation and long term culturing. In comparison to both other techniques this procedure is faster if direct preparation is used. Results can be obtained even on the same day. The main disadvantage, however, is the problem with the reliability of the direct preparation results. They must be confirmed by time-consuming long-term culturing. Data are presented on the likelihood of abnormal ultrasound findings being caused by chromosomal aberrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Amniocentesis↗

PubMed1995

The human protein kinase gene PKX1 on Xp22.3 displays Xp/Yp homology and is a site of chromosomal instability.

We have isolated a gene, PKX1, by virtue of its position within the candidate region for chondrodysplasia punctata in Xp22.3. Although data from one patient render it unlikely that PKX1 is the CDPX gene, this gene shows several interesting features. First, PKX1 appears to encode a novel type of human protein kinase that is related to the catalytic subunit of cAMP-dependent protein kinases and has striking homology to the DC2 protein kinase from Drosophila melanogaster. Second, PKX1 is part of a family of at least four genes or pseudogenes, of which three map to the human sex chromosomes. In contrast to all other genes from the X-specific region of Xp22.3, PKX1 has a homologue on Yp rather than Yq. This is intriguing as it indicates that the single pericentric inversion event hypothesized to have occurred during primate evolution is not sufficient to explain the present X/Y-homology pattern of Xp22.3. Third, we have characterized patients with different chromosomal rearrangements in Xp22.3 or Yp and show that a high proportion of these have occurred within the PKX1 locus. This suggests that the PKX1 gene, besides harbouring a previously described hot-spot for illegitimate Xp/Yp-recombination, contains additional sequences predisposing to chromosomal breakage events.

Amino Acid Sequence↗

PubMed1994

Characterization of a microdissection library from human chromosome region 3p14.

Structural alterations in human chromosome region 3p14-p23 resulting in the inactivation of one or more tumor suppressor genes are thought to play a pathogenic role in small cell lung cancer, renal cell carcinoma, and other human neoplasms. To identify putative tumor suppressor genes, 428 recombinant clones from a microdissection library specific for human chromosome region 3p14 were isolated and characterized. Ninety-six of these (22.5%) were human single-copy DNA sequences, 57 of which were unique sequence clones. Forty-four of these were mapped to the microdissected region using a cell hybrid mapping panel. Within this mapping panel, four probes detected two new chromosome breakpoints that were previously indistinguishable from the translocation breakpoint t(3;8) in 3p14.2 in hereditary renal cell carcinoma. One probe maps to the homozygously deleted region of the small cell lung cancer cell line U2020. In addition, microdissection clones have been shown to be suitable for isolation of yeast artificial chromosomes.

Animals↗

PubMed1994

Six years' experience with rapid karyotyping in prenatal diagnosis: correlations between phenotype detected by ultrasound and fetal karyotype.

From September 1985 to March 1992, 804 amniotic fluid samples from 64 different diagnostic centres of the Federal Republic of Germany were sent to our laboratory exclusively for rapid karyotyping. The average time needed for notification of the analysed karyotype was 4.65 days when the 'pipette method' was used for chromosome harvesting and 5.97 days when the 'in situ' technique was used. The overall incidence of chromosome aberrations was 15.3 per cent. Data are presented about the likelihood of abnormal ultrasound findings being caused by chromosome aberrations. These findings include polyhydramnios, oligohydramnios, growth retardation, fetal effusions, neural tube defects, craniofacial defects, heart defects, gastroschisis and omphalocele, gastrointestinal tract defects, urinogenital defects, and limb defects. In future, such data need to contain larger numbers of cases for each week of gestation. This will improve the risk evaluation for each case with abnormal ultrasound findings, which should lead to better management during pregnancy, delivery, and postnatal care for those who require rapid karyotyping.

Amniocentesis↗

PubMed1994

The origin of human chromosome 2 analyzed by comparative chromosome mapping with a DNA microlibrary.

Fluorescence in situ hybridization (FISH) of microlibraries established from distinct chromosome subregions can test the evolutionary conservation of chromosome bands as well as chromosomal rearrangements that occurred during primate evolution and will help to clarify phylogenetic relationships. We used a DNA library established by microdissection and microcloning from the entire long arm of human chromosome 2 for fluorescence in situ hybridization and comparative mapping of the chromosomes of human, great apes (Pan troglodytes, Pan paniscus, Gorilla gorilla, Pongo pygmaeus) and Old World monkeys (Macaca fuscata and Cercopithecus aethiops). Inversions were found in the pericentric region of the primate chromosome 2p homologs in great apes, and the hybridization pattern demonstrates the known phylogenetically derived telomere fusion in the line that leads to human chromosome 2. The hybridization of the 2q microlibrary to chromosomes of Old World monkeys gave a different pattern from that in the gorilla and the orang-utan, but a pattern similar to that of chimpanzees. This suggests convergence of chromosomal rearrangements in different phylogenetic lines.

Animals↗