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Biomedical subjects

R Siebert

Publications and source records attributed to R Siebert.

At least 91 records · Page 5Linked to original sources
PubMed1998

Application of interphase fluorescence in situ Hybridization for the detection of the Burkitt translocation t(8;14)(q24;q32) in B-cell lymphomas.

The translocation t(8;14)(q24;q32) is the characteristic chromosomal aberration of Burkitt's-type lymphomas and leukemias (BLs). On the molecular level, the t(8;14) juxtaposes the c-myc gene in 8q24 next to the IgH locus in 14q32, resulting in overexpression of the transcription factor c-Myc. The detection of a t(8;14) is a major aim in the diagnostic process of all patients with high-grade B-cell lymphomas because treatment strategies differ between BL and other high-grade lymphomas. As chromosome analyses are sometimes hampered by the low yield or poor quality of metaphase spreads and as the application of molecular genetic techniques is limited by the distribution of the 8q24 breakpoints over a region of about some hundred kilobases, we set out to establish an interphase fluorescence in situ hybridization (FISH) assay for the detection of the t(8;14). A cosmid probe hybridizing to the IgH constant region in 14q32 was combined with a differently labeled probe of pooled cosmid clones spanning the c-myc locus in 8q24. Interphase nuclei lacking a t(8;14) show two separated signals corresponding to each probe, whereas interphase nuclei carrying a t(8;14) display a split of the c-myc probe and a colocalization of at least one of the splitted signals with the IgH probe. Based on the results of extensive control studies, the cutoff level for this stringent (type I) criteria was set at 2%. Additionally, colocalization of at least one c-myc signal with one IgH signal alone (without signal split for the c-myc probe) was used as a less stringent (type II) criteria with a cutoff limit of 11%. Nine BLs and one Burkitt-like lymphoma were investigated by this approach. Cytogenetically, all tumors contained a translocation t(8;14)(q24;q32) except for one BL, in which cytogenetic analysis had failed. In interphase FISH, all lymphomas and leukemias met the less stringent criteria for the diagnosis of the t(8;14). Additionally, in all tumors but the Burkitt-like lymphoma, a t(8;14) could be diagnosed according to the stringent criteria. The percentage of cells found to harbor the t(8;14) by FISH ranged from 4.3% to 100%. Comparison of cytogenetic and FISH results revealed a significantly lower percentage of t(8;14)+ interphase nuclei than metaphase cells (P = .004). In conclusion, the described FISH assay provides a feasible and sensitive tool for the routine detection of the translocation t(8;14) in interphase cells which might also offer new insights into the biology of high-grade B-cell lymphomas.

Adult↗
PubMed1998

Chromosomal abnormalities in nodal and extranodal CD30+ anaplastic large cell lymphomas: infrequent detection of the t(2;5) in extranodal lymphomas.

To determine the significance of the t(2;5)(p23;q35) translocation in nodal and extranodal anaplastic large cell lymphoma (ALCL), we performed cytogenetic, molecular genetic, and immunohistochemical analyses of tumor tissues from 11 patients with CD30+ ALCL. Three of five patients with nodal ALCL had additional infiltration of the skin. Six patients had extranodal ALCL, two had primary intestinal ALCL, three had a primary cutaneous ALCL, and one had osseous ALCL. Cytogenetic investigation detected the t(2;5) in all patients with nodal ALCL but not extranodal ALCL. Tumor cells in t(2;5)+ lesions also stained immunohistochemically for p80NPM/ALK, whereas no staining for p80NPM/ALK was detected in extranodal ALCL. Two extranodal lesions had NPM/ALK fusion transcripts detected by nested reverse transcriptase-polymerase chain reaction. Fluorescence in situ hybridization analysis of these two lymphomas showed in one case a significant number (4%) of cells with a split hybridization signal, indicative of disruption of the NPM gene. Additional recurrent breakpoints observed in extranodal ALCL were 1p36, 6p25, and 8q24. Loss of genetic material occurred at 6q in one extranodal ALCL. Our results suggest that the t(2;5) more frequently plays a pathogenetic role in primary nodal than in extranodal ALCL and that this translocation may not be the primary event in some CD30+ ALCL.

Adult↗
PubMed1998

Detection of 6q deletions in breast carcinoma cell lines by fluorescence in situ hybridization.

Dual-color fluorescence in situ hybridization was performed to detect the frequency and extent of 6q deletions in ten breast carcinoma cell lines. In five cell lines, the 6q deletions involved large regions extending from 6q12-q16 to 6q27, and in one the deletion extended from the region distal to YAC 751G10 at 6q25.1 to 6q27. In two cell lines, 6q deletions occurred only in cells with polysomy 6, indicating that such deletions might be secondary chromosomal aberrations and reflect late genetic changes in breast carcinomas. In addition, an overrepresentation of 6q21-q22.2 was detected in one cell line.

Aneuploidy↗
PubMed1998

Application of interphase cytogenetics for the detection of t(11;14)(q13;q32) in mantle cell lymphomas.

BACKGROUND: The chromosomal translocation t(11;14)(q13;q32) is the hallmark of mantle cell lymphoma (MCL) in which it can be detected cytogenetically in about 75% of cases. The t(11;14) translocation juxtaposes the bcl-1 locus in chromosome band 11q13 next to the IgH locus in chromosome band 14q32 and, thus, leads to deregulation of the cell cycle regulatory protein cyclin D1, which is encoded by the CCND1 gene localized at the telomeric border of the bcl-1-locus. MCL has the worst prognosis of all low-grade non-Hodgkin's lymphomas (NHL). In some instances, however, histopathologic differentiation between MCL and other low-grade B-cell NHL is difficult. Therefore, detection of the t(11;14) translocation is of essential diagnostic value for the risk-adjusted management of patients with MCL. Unfortunately, chromosome analyses are frequently hampered by the low yield and quality of tumor metaphases. As the 11q13 breakpoints are scattered over a region of more than 120 kb the application of molecular genetic techniques is also limited. PATIENTS AND METHODS: We established an interphase fluorescence in situ hybridization (FISH) approach for the detection of the t(11;14) translocation by use of a cosmid probe hybridizing to the IgH constant region and a YAC spanning the bcl-1 region. Cells containing a t(11;14) translocation show a colocalisation of the signals for IgH and bcl-1. Eight control samples and 15 MCL specimens were investigated. RESULTS: According to our control studies, samples containing more than 10% of cells with this signal constellation can be diagnosed as carrying a clonal t(11;14) translocation. All eleven MCL found to carry the t(11;14) translocation by chromosome analysis were positive in our FISH assay. Additionally, two of four MCL lacking a clonal t(11;14) translocation by chromosome analysis were shown to carry this aberration in 14% and 37% of interphase nuclei. Southern blot data indicate that our FISH assay reliably detects the t(11;14) translocation irrespective of the location of the breakpoints within the bcl-1 region. CONCLUSIONS: The described interphase FISH assay provides a reliable and routinely applicable tool for diagnosis of the t(11;14) translocation.

Aged↗
PubMed1998

Detection of deletions in the short arm of chromosome 3 in uncultured renal cell carcinomas by interphase cytogenetics.

PURPOSE: Analysis of genetic alterations may facilitate the differential diagnosis of renal cell carcinoma (RCC) subtypes. For genetic classification, deletion of the short arm of chromosome 3 (3p), the hallmark of nonpapillary/clear cell RCC, is a major diagnostic criterion. Because of the limited routine applicability of cytogenetics and molecular genetic techniques we investigated interphase fluorescence in situ hybridization (FISH) for the detection of this aberration in RCC. MATERIALS AND METHODS: Using seven chromosome 3 specific probes FISH was performed on isolated nuclei from 26 uncultured sporadic RCC. RESULTS: Alterations of chromosome 3 were identified in 19 RCC (73%). Monosomy and/or 3p-deletions were observed in 15 of 19 (79%) non-papillary/clear cell RCC but not in other morphologic subgroups. The median percentage of cells in a specimen containing loss of 3p was 45%. Deletion mapping indicated that large deletions affecting different regions in 3p are predominant. Chromosomal region 3p24 was recurrently involved in all RCC with a deletion in 3p. CONCLUSION: Interphase FISH for the detection of loss in 3p provides a sensitive and feasible method for the genetic classification of kidney tumors and the delineation of recurrently deleted regions in 3p.

Adult↗
PubMed1997

Mapping of the gene encoding human beta-defensin-2 (DEFB2) to chromosome region 8p22-p23.1.

We recently reported the isolation of human beta-defensin-2 (hBD-2), a novel epithelia-derived peptide antibiotic belonging to the beta-defensin family. hBD-2 is expressed in skin and epithelia of the airway system, where it is believed to contribute to its antimicrobial defense. By fluorescence in situ hybridization using a hBD-2 genomic DNA probe and subsequent fluorescence R-banding, the hBD-2 gene (HGMW-approved symbol DEFB2) was assigned to human chromosome region 8p22-p23.1. PCR with a set of CEPH YAC clones spanning this chromosomal region revealed CEPH YACs 773G4, 920D12, and 820B4 to contain the hBD-2 gene. Relying on the preexisting physical maps of 8p22-p23.1, the hBD-2 gene was mapped in close proximity to D8S1993 (WI-9956) within the interval flanked by D8S552 and D8S1130 (CHLC.GATA25C10). The fact that all currently described genes encoding defensins map to chromosome 8p21-pter suggests that a gene cluster in this chromosomal region may play a major role in antimicrobial defense.

Blood Proteins↗
PubMed1997

Frequent deletions of 6q23-24 in B-cell non-Hodgkin's lymphomas detected by fluorescence in situ hybridization.

Deletions of the long arm of chromosome 6 (6q) are among the most frequent chromosome aberrations in malignant lymphomas and often occur as secondary changes in addition to typical translocations, such as t(14;18). Using fluorescence in situ hybridization (FISH) with two YAC probes hybridizing to 6q23-24 and with the centromeric probe D6Z1 as internal control, we studied 31 cases of low-grade and eight cases of high-grade B-cell lymphoma. Deletions of 6q23-24 were detected in 21 patients (56.8%) by FISH, compared to 13 patients (33.3%) by chromosome analysis. Deletions of 6q23-24 were found by FISH in 5 of 13 cases of small lymphocytic lymphoma, in 2 of 3 cases of mantle cell lymphoma, in 10 of 14 cases of t(14;18) positive low-grade follicular lymphoma, and in 4 of 8 cases of high-grade follicular lymphoma. This study shows that deletions of 6q23-24 are more frequent in B-cell lymphomas than previously suggested and that they can be detected more sensitively by FISH than by chromosome analysis. Contrary to previous reports indicating that the region 6q23-24 is preferentially deleted in low-grade lymphomas without t(14;18), our results indicate that deletions of 6q23-24 appear to be common in other pathological subsets of B-cell lymphoma as well, especially in follicular lymphomas with t(14;18).

Centromere↗
PubMed1997

Familial Hodgkin's and non-Hodgkin's lymphoma: different patterns in first-degree relatives.

In order to evaluate the importance of genetic susceptibility in Hodgkin's disease (HD) and non-Hodgkin's lymphoma (NHL) we retrospectively analysed 31 families with lymphoma in first-degree relatives containing a total of 65 affected persons. We observed 20 HD/HD, 8 NHL/HD and 8 NHL/NHL pairs with median ages of diagnosis of 27, 36 and 48 years, respectively (p < 0.001). In HD/HD sib pairs were predominant in contrast to parent/child pairs in NHL/NHL (p = 0.04). There was a higher frequency of diseases with impaired immune function in NHL/NHL than in other pairs (p = 0.01). Comparison of ages and times of incidence of the pairs as well as sex-concordance rates are consistent with an age-specific genetic susceptibility to HD, but suggest a time-specific exposure in some NHL-prone families with or without compromised immune function.

Adolescent↗
PubMed1997

Recurrence of Hodgkin's disease after 10 or more years: late relapse or de-novo malignancy due to HLA-DPB1*0301-linked susceptibility?

Recurrences of Hodgkin's disease (HD) ten or more years after initial therapy are rare and heterogeneous concerning pathological, biological and clinical features. Though usually regarded as relapses of initial disease at least part of these late recurrences may represent de-novo HD due to an increased constitutional risk. Following recent reports genetic risk for HD may be linked to the HLA-DPB1*0301 allele. Therefore, we investigated DPB1 and other HLA class I and II gene loci in three patients with very late recurrences of HD presenting at our institution within the last two years. All patients carry the HD susceptibility allele HLA-DPB1*0301. The expected probability of three patients with HD displaying the HLA-DPB1*0301 phenotype by chance is only p = 0.022. As serologic investigations also revealed Epstein-Barr virus (EBV) activity in all three cases our results support a role of genetic susceptibility possibly leading to impaired immune responses to EBV in very late recurring HD. Additionally, HLA-DPB1*0301 may be valuable for identifying patients with HD who might be candidates for a long term follow-up.

Adolescent↗
PubMed1996

High incidence of chromosomal imbalances and gene amplifications in the classical follicular variant of follicle center lymphoma.

The classical follicular variant of follicle center lymphoma (FCL-fo) is associated with the chromosomal translocation t(14;18)(q32;q21). However, the sole presence of this translocation is not sufficient for malignant transformation, as demonstrated by experiments in a transgenic mouse model. Most of the secondary changes, which play a central role in tumor development and progression and which are presumed to be of prognostic value, are gains and losses of chromosomal material. We analyzed 28 FCL-fo patients using comparative genomic hybridization (CGH). The most frequent imbalances were gains on chromosomes X, 7, 8, 12, and 18 as well as losses of material on chromosome arm 6q. For chromosomes X, 8, 12, and 18, the CGH data allowed further narrowing of the relevant subregions. In addition, novel high-level DNA amplifications were identified in five instances mapping to chromosome bands 1p36, 6p21, 8q24 (2 patients), and 12q13-14. Previously, such amplifications have been identified very rarely in lymphomas. In the 2 patients with amplifications mapping to chromosomal band 8q24, involvement of the MYC proto-oncogene in the amplification unit was demonstrated by Southern blot analysis. These data provide further entry points for studies to identify genes relevant for tumor progression in FCL-fo.

Adult↗
PubMed1996

A numerical study of persistence length effects on DNA conformation in sequencing electrophoresis.

We have developed a program to evaluate the influence of DNA stiffness on molecular mobility and conformation during electrophoresis. This (currently) two-dimensional numerical study models DNA as a chain of uniformly charged beads connected to one another by elastic segments, of finite mean size, in the presence of fixed obstacles representing gel fibers. Contrary to the standard biased reptation model (BRM), our Langevin-type dynamics for the chain are microscopic and warrant studies of fine effects such as inner chain orientation. Using this model, we show that the introduction of a persistence length decreases the (saturated) mobility at high electric fields, providing strong evidence that the gel generates a friction force and not only a (dissipation-free) constraint force. We also show that the persistence length leads to an increase of the chain orientation in the field direction. This suggests that DNA stiffness causes the saturation plateau value to be reached for smaller chain sizes than those predicted by the BRM model.

DNA↗
PubMed1996

Analysis of the novel cyclin-dependent kinase 4 and 6 inhibitor gene p18 in lymphoma and leukemia cell lines.

The genes for the CDK4/6-inhibitors p16INK4A/MTS1 and p15INK4B/MTS2 are frequently deleted in hematological malignancies. A new member of this family of CDK4/6 inhibitors is p18. In order to assess p18 growth-suppressor gene alterations in hematological neoplasms, we investigated 31 lymphoma and leukemia cell lines by PCR for both exons of this gene. No homozygous deletions were observed. Investigation of a new intragenic restriction fragment length polymorphism revealed no differences in allele distribution between the tumor cell lines and healthy volunteers. Our results suggest that homozygous deletion of the p18 gene does not play a major role in leukemogenesis or lymphomagenesis.

Base Sequence↗
PubMed1996

Genetic instability of 3p12-p21-specific microsatellite sequences in renal cell carcinoma.

OBJECTIVE: To determine the role of structural alterations of human chromosome region 3p12-p21 in the possible inactivation of one or more tumour-suppressor genes in the pathogenesis of renal cell carcinoma (RCC), lung cancer and other neoplasms. MATERIALS AND METHODS: As microsatellite instability (MI), in particular MI with loss of heterozygosity (LOH), may indicate putative tumour-suppressor gene loci, 20 kidney tumours, including 14 clear cell carcinomas and six non-clear cell neoplasms, were investigated with 10 polymorphic simple sequence-repeat markers spanning 3p12-p21. Six of these markers map to the region of deletion flanked by markers D3S1285 and D3S1295 bracketing the t(3;8) translocation break-point in 3p14.2 of hereditary RCC. RESULTS: Twelve of 14 clear cell RCCs displayed MI for at least one locus, as opposed to none of the non-clear cell tumours (P = 0.001). Locus D3S1274 in 3p13 located in the region deleted in lung cancer line U2020 and loci D3S1313 and D3S1300 in 3p14.3 characterized common regions of instability and LOH. Two patients with RCC who also had lung cancer and colon cancer, respectively, showed LOH at D3S1313 or D3S1300 as the only alterations of their kidney tumours. CONCLUSIONS: These results suggest that human chromosome region 3p14.3 distal to the hereditary t(3;8) translocation breakpoint and the region deleted in the U2020 lung cancer cell line might be involved in the tumorigenesis or progression of clear cell RCC.

Adult↗
PubMed1996

Construction of a consistent YAC contig for human chromosome region 3p14.1.

Chromosomal deletions and translocations of human chromosome region 3p14 are observed in various human malignancies and suggest the existence of a tumor suppressor gene locus within this region. Tumors most frequently affected by these aberrations are small-cell lung cancer and renal-cell carcinoma. In continuation of our previously published YAC contig of chromosome region 3p14.2-p14.3, we report here on the construction of a YAC contig of at least 11 Mb that consisted of 171 YACs and covers the entire subregion 3p14.1. This contig includes the t(3;8) breakpoint of a hereditary renal-cell carcinoma localized in 3p14.2 and extends into human chromosome region 3p12-p13. It defines the order of 34 DNA probes in relation to reference markers D3S6 and D3S30 as well as the human protein tyrosine phosphatase-gamma gene. For 31 DNA probes we identified nonchimeric YACs by fluorescence in situ hybridization. The minimal tilling pathway consists of 16 yeast artificial chromosomes. As a prerequisite for identification of a putative tumor suppressor gene within this region, this contig renders human chromosome region 3p14.1 accessible to gene isolation.

Blotting, Southern↗