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1.
J Pathol ; 216(3): 307-16, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18773450

ABSTRACT

Most cancer genomes are characterized by the gain or loss of copies of some sequences through deletion, amplification or unbalanced translocations. Delineating and quantifying these changes is important in understanding the initiation and progression of cancer, in identifying novel therapeutic targets, and in the diagnosis and prognosis of individual patients. Conventional methods for measuring copy-number are limited in their ability to analyse large numbers of loci, in their dynamic range and accuracy, or in their ability to analyse small or degraded samples. This latter limitation makes it difficult to access the wealth of fixed, archived material present in clinical collections, and also impairs our ability to analyse small numbers of selected cells from biopsies. Molecular copy-number counting (MCC), a digital PCR technique, has been used to delineate a non-reciprocal translocation using good quality DNA from a renal carcinoma cell line. We now demonstrate microMCC, an adaptation of MCC which allows the precise assessment of copy number variation over a significant dynamic range, in template DNA extracted from formalin-fixed paraffin-embedded clinical biopsies. Further, microMCC can accurately measure copy number variation at multiple loci, even when applied to picogram quantities of grossly degraded DNA extracted after laser capture microdissection of fixed specimens. Finally, we demonstrate the power of microMCC to precisely interrogate cancer genomes, in a way not currently feasible with other methodologies, by defining the position of a junction between an amplified and non-amplified genomic segment in a bronchial carcinoma. This has tremendous potential for the exploitation of archived resources for high-resolution targeted cancer genomics and in the future for interrogating multiple loci in cancer diagnostics or prognostics.


Subject(s)
DNA, Neoplasm/genetics , Gene Dosage , Neoplasms/genetics , Polymerase Chain Reaction/methods , Carcinoma, Bronchogenic/genetics , DNA Primers/genetics , Gene Amplification , Genetic Markers , Genome, Human , Humans , Lung Neoplasms/genetics , Microdissection , Neoplasms/pathology , Paraffin Embedding , Tissue Fixation
2.
Genes Chromosomes Cancer ; 46(9): 852-60, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17592619

ABSTRACT

Patients who receive curative treatment for lung cancer can develop additional lung tumors that may or may not be related to the original tumor and thus require different clinical management. If a subsequent tumor has a pattern of allele loss, revealed by allelotype analysis, overlapping that of the first tumor, it is believed to be a local recurrence or metastasis. In this case history, we present loss of heterozygosity analyses of the original primary tumor, and two second primary tumors occurring in the ipsilateral and the contra-lateral lungs. The allelotyping suggests that these tumors are all clonally related but concordance is not complete. Our interpretation is that the original primary tumor and the two new primary tumors have developed to full malignancy independently, but are clonally related, possibly via a clone of motile progenitor cells. Deletion mapping of DNA from biopsies of this patient delineated a region in 4p16 that we had previously shown to be lost in the transition from carcinoma in situ to invasive tumor. We identified a minimally deleted region encompassing six genes including two candidate tumor suppressor genes, CRMP1 a lung cancer metastasis-suppressing gene and PPP2R2C a gene for a regulatory subunit of the PP2 complex known to suppress tumorigenesis, particularly viral induced transformation.


Subject(s)
Carcinoma in Situ/genetics , Chromosomes, Human, Pair 4/genetics , Loss of Heterozygosity , Lung Neoplasms/genetics , Carcinoma in Situ/pathology , DNA, Neoplasm/genetics , Humans , Lung Neoplasms/pathology , Neoplasm Invasiveness , Nerve Tissue Proteins/genetics , Phosphoprotein Phosphatases/genetics , Protein Phosphatase 2
3.
Thorax ; 58(3): 266-71, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12612310

ABSTRACT

Recent developments in the detection of pre-invasive lesions of the large airways by fluorescence bronchoscopy are reviewed and the clinical and biological implications discussed.


Subject(s)
Bronchoscopy/methods , Carcinoma in Situ/diagnosis , Carcinoma, Squamous Cell/diagnosis , Lung Neoplasms/diagnosis , Fluorescence , Forecasting , Humans
4.
Proc Natl Acad Sci U S A ; 98(26): 15062-6, 2001 Dec 18.
Article in English | MEDLINE | ID: mdl-11734623

ABSTRACT

Chromosome 3 allele loss in preinvasive bronchial abnormalities and carcinogen-exposed, histologically normal bronchial epithelium indicates that it is an early, possibly the first, somatic genetic change in lung tumor development. Candidate tumor suppressor genes have been isolated from within distinct 3p regions implicated by heterozygous and homozygous allele loss. We have proposed that DUTT1, nested within homozygously deleted regions at 3p12-13, is the tumor suppressor gene that deletion-mapping and tumor suppression assays indicate is located in proximal 3p. The same gene, ROBO1 (accession number ), was independently isolated as the human homologue of the Drosophila gene, Roundabout. The gene, coding for a receptor with a domain structure of the neural-cell adhesion molecule family, is widely expressed and has been implicated in the guidance and migration of axons, myoblasts, and leukocytes in vertebrates. A deleted form of the gene, which mimics a naturally occurring, tumor-associated human homozygous deletion of exon 2 of DUTT1/ROBO1, was introduced into the mouse germ line. Mice homozygous for this targeted mutation, which eliminates the first Ig domain of Dutt1/Robo1, frequently die at birth of respiratory failure because of delayed lung maturation. Lungs from these mice have reduced air spaces and increased mesenchyme, features that are present some days before birth. Survivors acquire extensive bronchial epithelial abnormalities including hyperplasia, providing evidence of a functional relationship between a 3p gene and the development of bronchial abnormalities associated with early lung cancer.


Subject(s)
Bronchi/pathology , Gene Deletion , Hyperplasia/genetics , Lung/embryology , Nerve Tissue Proteins/genetics , Receptors, Immunologic/genetics , Amino Acid Sequence , Animals , Epithelium/pathology , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Phenotype , Roundabout Proteins
5.
Semin Cancer Biol ; 9(4): 255-65, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10448113

ABSTRACT

In an attempt to improve the poor survival rates for lung cancer, therapeutic strategies require a deeper understanding of the biological events contributing to the formation and progression of the disease. In particular, the importance of studying the different stages of lung cancer including early pre-neoplasia is being recognised and studies examining genetic changes in pre-invasive and invasive lesions are being used to identify key events in tumorigenesis.


Subject(s)
Lung Neoplasms/genetics , Lung Neoplasms/pathology , Animals , Biomarkers, Tumor/analysis , Biomarkers, Tumor/genetics , Bronchi/pathology , Disease Progression , Humans , Lung Neoplasms/diagnosis , Lung Neoplasms/therapy , Neoplasm Invasiveness , Precancerous Conditions/genetics , Precancerous Conditions/pathology
6.
Cancer Genet Cytogenet ; 111(2): 105-10, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10347545

ABSTRACT

Homozygous deletions in tumor cells have been useful in the localization and validation of tumor suppressor genes. We have described a homozygous deletion in a lung cancer cell line (U2020) which is located within the most proximal of the three regions on the short arm of chromosome 3 believed to be lost in lung cancer development. Construction of a YAC contig map indicates that the deletion spans around 8 Mb, but no large deletion was apparent on conventional cytogenetic analysis of the cell line. To investigate this paradox, whole chromosome, arm-specific, and regional paints have been used. This analysis has revealed that genetic loss has occurred by complex rearrangements of chromosomes 3, rather than simple interstitial deletion. These studies emphasize the power of molecular cytogenetics to disclose unsuspected tumor-specific translocations within the extremely complex karyotypes characteristic of solid tumors.


Subject(s)
Carcinoma, Small Cell/genetics , Chromosome Deletion , Chromosomes, Human, Pair 3 , Lung Neoplasms/genetics , Chromosome Painting , Chromosomes, Artificial, Yeast , Chromosomes, Human , Homozygote , Humans , In Situ Hybridization , Translocation, Genetic , Tumor Cells, Cultured
7.
Cancer Genet Cytogenet ; 108(2): 110-9, 1999 Jan 15.
Article in English | MEDLINE | ID: mdl-9973937

ABSTRACT

Until recently the ability to analyze complex karyotypic rearrangements was totally dependent upon light microscopy of G-banded chromosomes. Developments in the area of molecular cytogenetics have revolutionized such analysis, making it possible to determine the nature of complex rearrangements. An extensive analysis has been made of the small cell lung carcinoma (SCLC) cell line U2020, using a combined approach of conventional and molecular cytogenetics, enabling a highly detailed karyotype to be constructed revealing rearrangements previously undetected by G-banding alone. This approach offers the opportunity to reassess other tumor karyotypes, particularly those of high complexity found in solid tumors, for tumor-specific consistent rearrangements indecipherable by conventional karyotyping.


Subject(s)
Carcinoma, Small Cell/genetics , Chromosome Aberrations , Lung Neoplasms/genetics , Carcinoma, Small Cell/pathology , Chromosome Banding , Chromosome Painting/methods , Humans , Karyotyping , Lung Neoplasms/pathology , Tumor Cells, Cultured
8.
Genes Chromosomes Cancer ; 21(4): 326-32, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9559344

ABSTRACT

RET is a receptor tyrosine kinase expressed in neuroendocrine cells and in tumors of these cell types. RET activation may be mediated by a ligand complex comprising glial cell line-derived neurotrophic factor (GDNF) and GDNF family receptor alpha-1 (GFR alpha-1). Activating RET mutations are found in the inherited cancer syndrome multiple endocrine neoplasia type 2 and in a subset of the related sporadic tumors, medullary thyroid carcinoma and pheochromocytoma, both being derived from neuroendocrine tissues. In one small study, mutations were identified in another tumor with neuroendocrine features, small cell lung carcinoma (SCLC). To determine whether RET mutations contribute to the pathogenesis of SCLC, we examined a panel of 54 SCLC cell lines. No mutations were identified in RET exons 10, 11, and 13-16, regions previously implicated in SCLC or other neuroendocrine tumors. We further examined the expression pattern of RET and the genes encoding the components of its ligand complex GDNF and GFR alpha-1, in 21 SCLC lines by using RT-PCR. Although we found no consistent pattern of expression for these three genes, RET was expressed in 57% of SCLC lines. Thus, although RET mutations appear unlikely to be an important step in the tumorigenesis of SCLC, the frequent expression of this gene suggests that RET may have a mitogenic role in a subset of SCLC cell lines.


Subject(s)
Carcinoma, Small Cell/genetics , Drosophila Proteins , Lung Neoplasms/genetics , Nerve Growth Factors , Nerve Tissue Proteins/genetics , Proto-Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/genetics , Carcinoma, Small Cell/metabolism , DNA Primers , DNA, Neoplasm/isolation & purification , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation, Neoplastic , Glial Cell Line-Derived Neurotrophic Factor , Glial Cell Line-Derived Neurotrophic Factor Receptors , Humans , Ligands , Lung Neoplasms/metabolism , Nerve Tissue Proteins/biosynthesis , Polymerase Chain Reaction , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins c-ret , RNA, Neoplasm/isolation & purification , Receptor Protein-Tyrosine Kinases/biosynthesis , Sequence Analysis, DNA , Tumor Cells, Cultured
9.
Cancer Res ; 56(7): 1609-14, 1996 Apr 01.
Article in English | MEDLINE | ID: mdl-8603409

ABSTRACT

Lung tumors, particularly squamous cell carcinomas, are believed to develop through a series of morphological abnormalities, driven by underlying somatic genetic changes. One way of studying this process is to analyze candidate somatic genetic changes in samples of squamous metaplasia and bronchial dysplasia of varying degrees of severity as well as tumor from the same patient. This assumes a clonal relationship between these lesions. In this article, we provide evidence that adjacent, physically distinct bronchial abnormalities are clonally related. This has been achieved using a plaque assay technique to detect the same p53 mutation, present throughout a tumor specimen, in a small proportion of cells in an adjacent squamous metaplasia. In addition, we have obtained two dysplasia samples from a tumor-free patient over a 9-month interval. The earlier sample had one p53 mutation, whereas the later sample has to p53 mutations on different alleles. Thus, the pattern of clonal evolution detected in the parallel samples mimics the pattern seen in longitudinal samples and supports the analysis of synchronously collected samples for the study of tumor progression.


Subject(s)
Lung Neoplasms/genetics , Base Sequence , Chromosome Deletion , DNA, Neoplasm/analysis , Genes, p53 , Humans , Molecular Sequence Data , Mutation , Polymorphism, Single-Stranded Conformational
10.
Genes Chromosomes Cancer ; 15(2): 102-7, 1996 Feb.
Article in English | MEDLINE | ID: mdl-8834173

ABSTRACT

Little is known about the biology of Merkel cell carcinoma (MCC), also called small cell carcinoma of the skin. MCC has similarities with small cell lung cancer (SCLC): both are neuroendocrine malignancies with early metastasis to distant sites and a poor prognosis. Small cell lung cancer biopsies are known to have frequent losses on chromosome 3 in the region 3p21, yet MCCs have not been reported to have 3p deletions by karyotypic analysis. Considering the similarities between SCLC and MCC, we investigated 26 MCC tumours for loss of heterozygosity (LOH) on 3p. First, RFLP analysis was performed using PCR with nine primer sets from six loci. Second, 25 tumours were examined by microsatellite analysis for 3p markers D3S1289 and D3S1285 and SST on 3q. All 26 tumours were informative at one or more loci; of these, 18 (69%) demonstrated LOH for at least one marker on the short arm. For all informative loci the frequency of LOH was greater than 30% (range 33-75%). In a cell line derived from one tumour, it was possible to demonstrate rearrangement of chromosome 3 by in situ hybridisation. No LOH was seen in 15 informative cases for the 3q locus SST. A region 3p13-p21.1, centered on the marker D3S2, was deleted in all tumours demonstrating LOH, with a secondary deletion involving D3S30 detected in some tumours at 3p13. Our results indicate that LOH on 3p is a common occurrence in MCC; however, three tumours for which DNA was also available from a corresponding cell line suggest there may be a subset of MCC whose genesis is independent of deletions of 3p.


Subject(s)
Carcinoma, Merkel Cell/genetics , Chromosomes, Human, Pair 3/genetics , Skin Neoplasms/genetics , Cell Line, Transformed , Chromosome Mapping , Chromosomes, Human, Pair 3/ultrastructure , DNA, Neoplasm/genetics , Herpesvirus 4, Human , Humans , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Sequence Deletion , Skin Neoplasms/ultrastructure , Tumor Cells, Cultured
11.
Oncogene ; 11(12): 2591-8, 1995 Dec 21.
Article in English | MEDLINE | ID: mdl-8545116

ABSTRACT

Epithelial tumours develop through a sequence of pre-invasive lesions of increasing disarray driven by underlying somatic genetic changes. We have studied the occurrence of the two most common somatic genetic changes associated with lung cancer in a series of premalignant bronchial lesions representing different stages in lung tumorigenesis. We present evidence that allele loss on chromosome 3 precedes damage to the p53 gene. Damage to chromosome 3 itself appears to be sequential in that the pattern of allele loss seen in dysplasia is often much more discrete than in invasive tumours. This implies that preneoplastic lesions may be a useful source of material for deletion mapping studies aimed at localising the position of tumour suppressor genes. We illustrate this by the comparison of an interstitial deletion described in this study with a homozygous deletion we have described previously, which has resulted in a better definition of the localisation of a tumour suppressor gene believed to be involved in lung cancer development.


Subject(s)
Chromosome Deletion , Chromosomes, Human, Pair 3 , Lung Neoplasms/genetics , Base Sequence , Genes, p53 , Humans , Molecular Sequence Data , Mutation
12.
Int J Cancer ; 58(6): 787-92, 1994 Sep 15.
Article in English | MEDLINE | ID: mdl-7927869

ABSTRACT

Loss of heterozygosity (LOH) has been extensively studied on the short arm of chromosome 3, and functional proofs have been obtained defining a tumor-suppressor locus at 3p21-22. We examined 31 paraffin-embedded cervical cancer samples for LOH, using 5 PCR-primer pairs, located around 3p21. Allele loss was found in 19 out of the 27 informative samples (70%) while 13 out of 23 informative samples (56%) had LOH located at 3p21-22. More of the human papillomavirus (HPV)-positive samples had LOH compared to the HPV-negative samples, giving only a weak association between loss of allele and HPV integration. Modifications of the DNA in the formaldehyde-fixed samples were detected, and further studies will be required to clarify how such artifacts may affect restriction fragment length polymorphism (RFLP) studies on fixed tissues.


Subject(s)
Chromosomes, Human, Pair 3 , DNA, Neoplasm/analysis , DNA, Neoplasm/genetics , Formaldehyde/pharmacology , Polymerase Chain Reaction/methods , Polymorphism, Restriction Fragment Length , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/pathology , Alleles , Base Sequence , Female , Gene Deletion , Genetic Variation , Humans , Molecular Sequence Data , Paraffin Embedding , Reproducibility of Results , Tissue Fixation
13.
Br Med Bull ; 50(3): 688-97, 1994 Jul.
Article in English | MEDLINE | ID: mdl-7987648

ABSTRACT

Both inherited predisposition and acquired somatic genetic changes contribute to lung tumour development but the former is likely to be the minor component. The relative ease with which lung tumours can be established as cell lines has resulted in extensive cytogenetic analysis, and, this together with molecular techniques which assess loss of genetic material has revealed consistent patterns of genetic damage. Some of these characteristic somatic genetic changes have been traced back to the pre-invasive stage of tumour development and this information may contribute to the future management of this currently intractable disease.


Subject(s)
Lung Neoplasms/genetics , Chromosome Deletion , Chromosomes, Human, Pair 3/genetics , Genes, Retinoblastoma , Humans , Lung Neoplasms/diagnosis , Oncogenes , Tumor Suppressor Protein p53/genetics
14.
Eur J Cancer ; 30A(11): 1705-9, 1994.
Article in English | MEDLINE | ID: mdl-7833148

ABSTRACT

In a number of cell lines with a multidrug resistant phenotype, there is no overexpression of the putative efflux pump, P-glycoprotein. Some such lines do, however, overexpress the MRP gene which encodes a protein bearing considerable amino acid homology to P-glycoprotein. We have used in situ hybridisation to study expression of the MRP gene in human cell lines, lung tumours (representing all the major histologies) and normal lung tissue. Considerable heterogeneity of expression was seen in parental cell line COR-L23/P whereas relatively uniform high-level expression was seen in the resistant line COR-L23/R. Normal bronchial epithelium was strongly positive, but the major epithelial component of all eight lung tumours studied showed only a negative to weak signal. However, the leading edge of the tumours consistently produced a more intense signal similar to that in normal epithelium. Areas of lymphocytic infiltrate were more strongly positive than the tumour epithelium. These results suggest that expression of the MRP gene may be a significant factor determining response of lung tumours to chemotherapy, but that considerable caution is needed in the interpretation of expression studies carried out on homogenised tissue biopsies.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Drug Resistance, Multiple/genetics , Lung Neoplasms/genetics , Base Sequence , Blotting, Northern , Gene Expression , Humans , In Situ Hybridization , Molecular Sequence Data , Oligonucleotide Probes , RNA, Messenger/genetics , RNA, Neoplasm/genetics , Tumor Cells, Cultured
15.
Cytogenet Cell Genet ; 66(1): 42-7, 1994.
Article in English | MEDLINE | ID: mdl-8275707

ABSTRACT

In constructing complete human chromosome maps, the relative order of markers and their precise chromosomal location will be combined. Multicolour in situ hybridisation, in which two probes are simultaneously hybridised to chromosomes and subsequently distinguished, potentially will provide both types of information. Using this technique, we have produced an ordered map of eight human chromosome 3 DNA markers, using small, single-copy probes that can detect target sequences ranging in size from 4 kb to as little as 500 bp.


Subject(s)
Chromosome Mapping/methods , Chromosomes, Human, Pair 3 , Chromosomes, Human , In Situ Hybridization, Fluorescence/methods , Lymphocytes/cytology , Cells, Cultured , Fluorescent Dyes , Humans , Indoles , Lymphocyte Activation , Lymphocytes/immunology , Metaphase
16.
J Natl Cancer Inst ; 86(2): 110-7, 1994 Jan 19.
Article in English | MEDLINE | ID: mdl-7903701

ABSTRACT

BACKGROUND: A 190k (190-kilodalton) membrane protein has been identified in several multidrug-resistant (MDR) cell lines that show decreased drug accumulation without expression of P-glycoprotein. It is not clear whether this 190k protein is involved directly in drug efflux. Recently, a gene for a putative transporter protein, MRP (multidrug resistance-associated protein) has been sequenced and localized to chromosome 16. The protein encoded by this gene contains a 7-amino-acid sequence present in the synthetic peptide used to generate the antiserum recognizing the 190k protein. PURPOSE: The study was undertaken to clarify the relationship of the 190k protein to MRP gene expression in non-P-glycoprotein-containing MDR cells of the large-cell and adenocarcinoma lung cancer lines, COR-L23 and MOR. METHODS: Expression of the 190k protein was determined by Western blot analysis and that of the MRP gene by polymerase chain reaction amplification of complementary DNA reverse transcribed from RNA. Abnormalities of chromosome 16 were investigated in chromosome spreads by fluorescence in situ hybridization. RESULTS: The amount of detectable 190k protein is closely associated with degree of drug resistance. Cell lines surviving in higher drug concentrations have greater amounts of protein, and revertant lines grown without drug for up to 28 weeks show reduced expression of the protein together with enhanced drug sensitivity. The 190k protein appears to be one of the major proteins differentially expressed in membranes of drug-resistant cells. The amount of MRP messenger RNA correlates closely with that of the 190k protein. The MDR cells contain amplified chromosome 16 material with many double minutes in the large-cell lung tumor lines and an enlarged chromosome 16 in the adenocarcinoma lines. CONCLUSION: The 190k protein detected immunologically is likely to be the protein, encoded by the MRP gene, which becomes overexpressed in these cells as a consequence of chromosomal amplification and fragmentation. IMPLICATION: Though associated with drug resistance, enhanced drug efflux, and decreased drug accumulation in cell lines, the role of this protein in clinical resistance has yet to be determined.


Subject(s)
Adenocarcinoma/genetics , Carcinoma, Large Cell/genetics , Carcinoma, Small Cell/genetics , Drug Resistance/genetics , Lung Neoplasms/genetics , Membrane Proteins/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1 , Adenocarcinoma/metabolism , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , Carcinoma, Large Cell/metabolism , Carcinoma, Small Cell/metabolism , Carrier Proteins , Chromosome Aberrations , Chromosome Disorders , Chromosomes, Human, Pair 16 , Drug Resistance/physiology , Gene Expression , Humans , In Situ Hybridization, Fluorescence , Lung Neoplasms/metabolism , Membrane Glycoproteins , Membrane Proteins/biosynthesis , Membrane Proteins/isolation & purification , Molecular Sequence Data , Polymerase Chain Reaction , Rabbits , Tumor Cells, Cultured
17.
Cytogenet Cell Genet ; 63(1): 64-5, 1993.
Article in English | MEDLINE | ID: mdl-8095447

ABSTRACT

We report here the characterization of a new polymorphic locus (D3S1207), shown by fluorescence in situ hybridization to map to 3p26-pter. The distal region of chromosome 3 has been implicated in both von Hippel-Lindau and 3p deletion syndrome, lung, uterine, breast, testicular, and ovarian cancers, renal cell and nasopharyngeal carcinomas, mesotheliomas, and various haematological malignancies, yet relatively few polymorphic marker probes are well mapped within this region. The identification of this new probe will therefore be particularly useful for the study of these diseases.


Subject(s)
Chromosome Deletion , Chromosomes, Human, Pair 3 , DNA Probes , Neoplasms/genetics , von Hippel-Lindau Disease/genetics , Blotting, Southern , Chromosome Mapping , Humans , Polymorphism, Restriction Fragment Length
18.
Hum Genet ; 90(1-2): 17-22, 1992.
Article in English | MEDLINE | ID: mdl-1358787

ABSTRACT

Genetic markers with high degrees of polymorphisms are of vital importance in the construction of high resolution (2-4 cM) linkage maps of human chromosomes as specified in the short-term goals of the Human Genome Initiative. In this paper, we report on molecular and genetic characterization and physical localization of 11 new multiallele restriction fragment length polymorphism markers on human chromosome 3p. Ten of these represent three- and four-allele polymorphisms of the base substitution type probably at two adjacent restriction sites. One has been identified as a novel mini-satellite sequence comprising a variable copy number tandem repeat array of a G/T-rich 79-bp sequence. This collection of multiallele polymorphic (PIC values: 0.40-0.60) markers should prove valuable and increase the resolution power of the available chromosome 3p genetic markers.


Subject(s)
Chromosomes, Human, Pair 3 , Genetic Markers/genetics , Polymorphism, Restriction Fragment Length , Alleles , Base Sequence , Female , Humans , In Situ Hybridization, Fluorescence , Male , Molecular Sequence Data , Pedigree , Polymerase Chain Reaction
19.
Genes Chromosomes Cancer ; 5(2): 119-27, 1992 Sep.
Article in English | MEDLINE | ID: mdl-1381947

ABSTRACT

Homozygous deletions are instrumental in the detection and cloning of tumor suppressor genes. We report the isolation and characterization of 39 new single-copy probes saturating a submicroscopic homozygous deletion detected in the DNA of the small cell lung cancer (SCLC) cell line U2020. The probes were selected from a large collection, covering the entire length of chromosome 3 with an estimated average spacing of 100-150 kb. Based on the number of probes in the deletion and the probe density, the size of the U2020 submicroscopic deletion was estimated to be in the range of 4-7 megabases. Among the deleted loci, 17 showed conservation across species, probably representing potential coding gene sequences. By genetic and physical mapping of a large randomly chosen fraction of the deleted probes, we defined the location of the U2020 deletion within chromosome band 3p12. Our cloning strategy is based on narrowing the region of interest by eliminating probes that retain heterozygosity in SCLC samples, thus selecting for probes in the region of common loss.


Subject(s)
Carcinoma, Small Cell/genetics , Chromosome Deletion , Chromosomes, Human, Pair 3 , Genes, Tumor Suppressor/genetics , Lung Neoplasms/genetics , Blotting, Southern , Cloning, Molecular , DNA Probes/genetics , Homozygote , Humans , Polymorphism, Restriction Fragment Length , Tumor Cells, Cultured
20.
Genes Chromosomes Cancer ; 5(1): 67-74, 1992 Jul.
Article in English | MEDLINE | ID: mdl-1384664

ABSTRACT

The small-cell lung carcinoma cell line U2020 contains a submicroscopic, homozygous deletion that removes a chromosomal segment within 3p13-p14, including the locus D3S3. We have sublocalized 49 additional probes to the 3p13-p14.2 region and have identified 7 new DNA markers that arise from within the U2020 deletion. The estimated size of the deletion, based on marker density, is approximately 4-5 megabases (Mb). Including D3S3, 7 of the 8 markers have been linked by pulsed-field gel (PFG) electrophoresis over an area of approximately 2 Mb. Including the one unlinked marker, PFG analysis accounts for about 3 Mb of the region. The U2020 deletion appears confined to the 3p13-p14.2 region and does not include the candidate tumor suppressor gene, protein-tyrosine phosphatase gamma (PTPG).


Subject(s)
Carcinoma, Small Cell/genetics , Chromosome Deletion , Chromosomes, Human, Pair 3/ultrastructure , Lung Neoplasms/genetics , Protein Tyrosine Phosphatases/genetics , Tumor Cells, Cultured , Carcinoma, Small Cell/pathology , DNA Probes , Electrophoresis, Gel, Pulsed-Field , Genetic Linkage , Genetic Markers , Humans , Lung Neoplasms/pathology
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