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PubMed2005

Genomic alterations detected by comparative genomic hybridization in primary lung adenocarcinomas with special reference to the relationship with DNA ploidy.

To clarify the clinicopathological and biological significance of genomic alterations in pulmonary adenocarcinomas, we examined chromosomal DNA sequence copy number aberrations (DSCNAs) and DNA ploidy in 42 surgically resected specimens by comparative genomic hybridization (CGH) and laser scanning cytometry (LSC), respectively. The number of DSCNA did not affect the size of carcinoma or number of nodal metastasis. More than 60% of carcinomas showed a 1q21-23 gain or 13q21 loss. Gains of 9q22-33 and 10q26-qter and a loss of 15q14-qter were significantly associated with nodal metastasis (p<0.05). Of 42 pulmonary adenocarcinomas, 32 (76%) showed DNA aneuploidy. The number of DSCNAs in aneuploid carcinomas was larger than that in diploid carcinomas (p<0.01). Our results suggest that diploid and aneuploid types are cytogenetically different in pulmonary adenocarcinomas and aneuploid carcinomas are genetically more unstable and aggressive than diploid carcinomas.

Adenocarcinoma↗
PubMed2002

Genomic imbalances detected by comparative genomic hybridization are prognostic markers in invasive ductal breast carcinomas.

AIMS: The aim of this work is the study of the prognostic significance of the chromosomal aberrations described in a series of invasive ductal breast carcinomas. METHODS AND RESULTS: We analysed by comparative genomic hybridization a group of 70 formalin-fixed paraffin-embedded invasive ductal breast carcinomas. Aberrations showed a frequency similar to previous studies using frozen tumours. Interestingly, we identified gains involving 6q16-q24 more frequently than in other series. We analysed the association among the chromosomal imbalances, 11 histopathological factors, relapse rate and overall survival of patients. Associations showed 16q losses as a potential marker of good prognosis, as they were more frequent in node-negative (P=0.025) and in oestrogen-positive tumours (P < 0.001). Furthermore, 100% of bcl-2+ tumours presented this aberration compared with 29.3% in bcl-2- (P=0.014). 1q, 11q, 17q and 20q gains were associated with poor prognosis: 95% of cases with 1q gains were bigger than 20 mm (P=0.041). Tumours with 1q and 11q gains showed a higher relapse rate (P=0.063; P=0.066). Within the good prognosis group of lymph node-negative patients, 17q and 20q gains identify a subgroup with increased relapse rate (P=0.039). CONCLUSIONS: Chromosomal imbalances, together with histopathological factors, may help to predict outcome in breast cancer patients.

Adult↗
PubMed1993

Detection of complete and partial chromosome gains and losses by comparative genomic in situ hybridization.

Comparative genomic in situ hybridization (CGH) provides a new possibility for searching genomes for imbalanced genetic material. Labeled genomic test DNA, prepared from clinical or tumor specimens, is mixed with differently labeled control DNA prepared from cells with normal chromosome complements. The mixed probe is used for chromosomal in situ suppression (CISS) hybridization to normal metaphase spreads (CGH-metaphase spreads). Hybridized test and control DNA sequences are detected via different fluorochromes, e.g., fluorescein isothiocyanate (FITC) and tetraethylrhodamine isothiocyanate (TRITC). The ratios of FITC/TRITC fluorescence intensities for each chromosome or chromosome segment should then reflect its relative copy number in the test genome compared with the control genome, e.g., 0.5 for monosomies, 1 for disomies, 1.5 for trisomies, etc. Initially, model experiments were designed to test the accuracy of fluorescence ratio measurements on single chromosomes. DNAs from up to five human chromosome-specific plasmid libraries were labeled with biotin and digoxigenin in different hapten proportions. Probe mixtures were used for CISS hybridization to normal human metaphase spreads and detected with FITC and TRITC. An epifluorescence microscope equipped with a cooled charge coupled device (CCD) camera was used for image acquisition. Procedures for fluorescence ratio measurements were developed on the basis of commercial image analysis software. For hapten ratios 4/1, 1/1 and 1/4, fluorescence ratio values measured for individual chromosomes could be used as a single reliable parameter for chromosome identification. Our findings indicate (1) a tight correlation of fluorescence ratio values with hapten ratios, and (2) the potential of fluorescence ratio measurements for multiple color chromosome painting. Subsequently, genomic test DNAs, prepared from a patient with Down syndrome, from blood of a patient with T-cell prolymphocytic leukemia, and from cultured cells of a renal papillary carcinoma cell line, were applied in CGH experiments. As expected, significant differences in the fluorescence ratios could be measured for chromosome types present in different copy numbers in these test genomes, including a trisomy of chromosome 21, the smallest autosome of the human complement. In addition, chromosome material involved in partial gains and losses of the different tumors could be mapped to their normal chromosome counterparts in CGH-metaphase spreads. An alternative and simpler evaluation procedure based on visual inspection of CCD images of CGH-metaphase spreads also yielded consistent results from several independent observers. Pitfalls, methodological improvements, and potential applications of CGH analyses are discussed.

Aneuploidy↗
PubMed2005

Web-based resources for comparative genomics.

The available web-based genome data and related resources provide great opportunities for biomedical scientists to identify functional elements in a particular genome region or to explore the evolutionary pattern of genome dynamics. Comparative genomics is an indispensable tool for achieving these goals. Because of the broad scope of comparative genomics, it is difficult to address all of its aspects in this short survey. A few currently 'hot' topics have therefore been selected and a brief review of the availability of web-based databases and software is given.

Animals↗
PubMed2006

Mapping Drosophila genomic aberration breakpoints with comparative genome hybridization on microarrays.

Chromosomal aberrations are genetic "reagents" that are commonly used in Drosophila research. Stocks containing chromosomes carrying large deletions of DNA (deficiency stocks, designated Df) as well as stocks carrying an extra copy of a chromosomal region (duplication stocks, designated Dp) are essential for a variety of genetic analyses. The extent of what is deleted or duplicated has typically been determined cytologically by salivary gland polytene chromosome squashes, which identify the edges of the aberration (so-called breakpoints) of each Df or Dp at low resolution. The margin of error for this technique can be quite high, however, because it is dependent on the quality of the squash and the experience of the scientist interpreting the data. Comparative genome hybridization on microarrays provides a precise molecular method to identify which regions of the genome are deleted or duplicated in these stocks by examining a change in chromosomal ploidy across the whole genome. Furthermore, this technique allows genetic data obtained with these strains to be placed in a molecular genomic context.

Animals↗
PubMed2005

Genomic changes identified by comparative genomic hybridisation in docetaxel-resistant breast cancer cell lines.

Docetaxel is one of the most effective chemotherapeutic agents in the treatment of breast cancer. Breast cancers can have an inherent or acquired resistance to docetaxel but the causes of this resistance remain unclear. In this study high-level, docetaxel-resistant human breast cancer cell lines (MCF-7 and MDA-MB-231) were created, and comparative genomic hybridisation was used to identify genomic regions associated with resistance to docetaxel. MCF-7 resistant cells showed an amplification of chromosomes 7q21.11-q22.1, 17q23-q24.3, 18, and deletion of chromosomes 6p, 10q11.2-qter and 12p. MDA-MB-231 resistant cells showed a gain of chromosomes 5p, 7q11.1-q35, 9, and loss of chromosomes 4, 8q24.1-qter, 10, 11q23.1-qter, 12q15-q24.31, 14q and 18. Whole chromosome paints confirmed these findings. Amplification of 7q21 and loss of 10q may represent a common mechanism of acquired docetaxel resistance in breast cancer cells. This study is the first description of a genomic approach specifically to identify genomic regions involved in resistance to docetaxel.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

OCGR array: an oral cancer genomic regional array for comparative genomic hybridization analysis.

Genetic alterations have been recognized as important events in the carcinogenesis of oral squamous cell carcinoma (OSCC) and have been used as predictors of progression risk. In this study, we have designed an oral cancer-specific human bacterial artificial chromosome (BAC) array, called the oral cancer genomic regional array (OCGR), to detect and fine map copy number alterations in OSCC. This array contains a total of approximately 45 Mbp coverage of nine chromosomal regions reported to be involved in the progression of oral cancer. We demonstrate the detection of copy number alterations in 14 microdissected clinical specimens in each of the nine regions. These include both copy number increases and decreases. Although the number of regions selected for this first generation array is small, we observed multiple segmental changes. In some cases, we observed single BAC clone alterations at 7p11 and 11q13 which contain EGFR and cyclin D1 respectively highlighting the need for high resolution detection techniques. Array comparative genomic hybridization (CGH) complements traditional methods for detecting genetic alterations in OSCC (such as microsatellite and CGH analysis) by improving the detection of segmental copy number alterations to single BAC clone resolution. This work represents the first attempt at the construction of an oral cancer-specific CGH array.

Acid Anhydride Hydrolases↗
PubMed1999

Comparative genomic hybridization, loss of heterozygosity, and DNA sequence analysis of single cells.

A PCR strategy is described for global amplification of DNA from a single eukaryotic cell that enables the comprehensive analysis of the whole genome. By comparative genomic hybridization, not only gross DNA copy number variations, such as monosomic X and trisomic 21 in single male cells and cells from Down's syndrome patients, respectively, but multiple deletions and amplifications characteristic for human tumor cells are reliably retrieved. As a model of heterogeneous cell populations exposed to selective pressure, we have studied single micrometastatic cells isolated from bone marrow of cancer patients. The observed congruent pattern of comparative genomic hybridization data, loss of heterozygosity, and mutations as detected by sequencing attests to the technique's fidelity and demonstrates its usefulness for assessing clonal evolution of genetic variants in complex populations.

Bone Marrow Cells↗
PubMed2003

Genome sequencing and comparative genomics of tropical disease pathogens.

The sequencing of eukaryotic genomes has lagged behind sequencing of organisms in the other domains of life, archae and bacteria, primarily due to their greater size and complexity. With recent advances in high-throughput technologies such as robotics and improved computational resources, the number of eukaryotic genome sequencing projects has increased significantly. Among these are a number of sequencing projects of tropical pathogens of medical and veterinary importance, many of which are responsible for causing widespread morbidity and mortality in peoples of developing countries. Uncovering the complete gene complement of these organisms is proving to be of immense value in the development of novel methods of parasite control, such as antiparasitic drugs and vaccines, as well as the development of new diagnostic tools. Combining pathogen genome sequences with the host and vector genome sequences is promising to be a robust method for the identification of host-pathogen interactions. Finally, comparative sequencing of related species, especially of organisms used as model systems in the study of the disease, is beginning to realize its potential in the identification of genes, and the evolutionary forces that shape the genes, that are involved in evasion of the host immune response.

Animals↗
PubMed2004

Genome-wide array-based comparative genomic hybridization of diffuse large B-cell lymphoma: comparison between CD5-positive and CD5-negative cases.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma and exhibits aggressive and heterogeneous clinical behavior. To genetically characterize DLBCL, we established our own array-based comparative genomic hybridization and analyzed a total of 70 cases [26 CD-positive (CD5+) DLBCL and 44 CD5-negative (CD5-) DLBCL cases]. Regions of genomic aberrations observed in >20% of cases of both the CD5+ and CD5- groups were gains of 1q21-q31, 1q32, 3p25-q29, 5p13, 6p21-p25, 7p22-q31, 8q24, 11q23-q24, 12q13-q21, 16p13, 18, and X and losses of 1p36, 3p14, 6q14-q25, 6q27, 9p21, and 17p11-p13. Because CD5 expression marks a subgroup with poor prognosis, we subsequently analyzed genomic gains and losses of CD5+ DLBCL compared with those of CD5-. Although both groups showed similar genomic patterns of gains and losses, gains of 10p14-p15 and 19q13 and losses of 1q43-q44 and 8p23 were found to be characteristic of CD5+ DLBCL. By focusing on the gain of 13q21-q34 and loss of 1p34-p36, we were also able to identify prognostically distinct subgroups among CD5+ DLBCL cases. These results suggest that array-based comparative genomic hybridization analysis provides a platform of genomic aberrations of DLBCL both common and specific to clinically distinct subgroups.

CD5 Antigens↗
PubMed2002

Vibrio cholerae phage K139: complete genome sequence and comparative genomics of related phages.

In this report, we characterize the complete genome sequence of the temperate phage K139, which morphologically belongs to the Myoviridae phage family (P2 and 186). The prophage genome consists of 33,106 bp, and the overall GC content is 48.9%. Forty-four open reading frames were identified. Homology analysis and motif search were used to assign possible functions for the genes, revealing a close relationship to P2-like phages. By Southern blot screening of a Vibrio cholerae strain collection, two highly K139-related phage sequences were detected in non-O1, non-O139 strains. Combinatorial PCR analysis revealed almost identical genome organizations. One region of variable gene content was identified and sequenced. Additionally, the tail fiber genes were analyzed, leading to the identification of putative host-specific sequence variations. Furthermore, a K139-encoded Dam methyltransferase was characterized.

Bacteriophages↗
PubMed2002

An orphan gyrB in the Mycobacterium smegmatis genome uncovered by comparative genomics.

DNA gyrase is an essential topoisomerase found in all bacteria. It is encoded by gyrB and gyrA genes. These genes are organized differently in different bacteria. Direct comparison of Mycobacterium tuberculosis and Mycobacterium smegmatis genomes reveals presence of an additional gyrB in M. smegmatis flanked by novel genes. Analysis of the amino acid sequence of GyrB from different organisms suggests that the orphan GyrB in M. smegmatis may have an important cellular role.

Amino Acid Sequence↗
PubMed2000

Comprehensive chromosomal analysis of human preimplantation embryos using whole genome amplification and single cell comparative genomic hybridization.

Analysis of small numbers of chromosomes using interphase fluorescent in-situ hybridization (FISH) probes has revealed that 50% of human preimplantation embryos contain abnormal cells. Detection of high levels of mosaicism with so few probes has led some researchers to extrapolate that a full analysis of all 23 pairs of chromosomes would reveal that all human embryos contain a proportion of abnormal cells. However, existing cytogenetic protocols cannot achieve such an analysis due to technical limitations. We have developed a novel technique based on whole genome amplification and comparative genomic hybridization (CGH), which for the first time allows the copy number of every chromosome to be assessed in almost every cell of a cleavage-stage embryo. We have successfully analysed 64 cells (blastomeres) derived from 12 embryos and have detected unusual forms of aneuploidy, high levels of chromosomal mosaicism, non-mosaic aneuploidy and chromosome breakage. This is the first report of a comprehensive assessment of chromosome copy number in human embryos and indicates that, despite high levels of mosaicism, some embryos do have normal chromosome numbers in every cell. Such embryos may have a superior developmental potential, and their low frequency may explain correspondingly low success rates of natural and assisted conception in humans.

Blastomeres↗
PubMed2006

CGHScan: finding variable regions using high-density microarray comparative genomic hybridization data.

BACKGROUND: Comparative genomic hybridization can rapidly identify chromosomal regions that vary between organisms and tissues. This technique has been applied to detecting differences between normal and cancerous tissues in eukaryotes as well as genomic variability in microbial strains and species. The density of oligonucleotide probes available on current microarray platforms is particularly well-suited for comparisons of organisms with smaller genomes like bacteria and yeast where an entire genome can be assayed on a single microarray with high resolution. Available methods for analyzing these experiments typically confine analyses to data from pre-defined annotated genome features, such as entire genes. Many of these methods are ill suited for datasets with the number of measurements typical of high-density microarrays. RESULTS: We present an algorithm for analyzing microarray hybridization data to aid identification of regions that vary between an unsequenced genome and a sequenced reference genome. The program, CGHScan, uses an iterative random walk approach integrating multi-layered significance testing to detect these regions from comparative genomic hybridization data. The algorithm tolerates a high level of noise in measurements of individual probe intensities and is relatively insensitive to the choice of method for normalizing probe intensity values and identifying probes that differ between samples. When applied to comparative genomic hybridization data from a published experiment, CGHScan identified eight of nine known deletions in a Brucella ovis strain as compared to Brucella melitensis. The same result was obtained using two different normalization methods and two different scores to classify data for individual probes as representing conserved or variable genomic regions. The undetected region is a small (58 base pair) deletion that is below the resolution of CGHScan given the array design employed in the study. CONCLUSION: CGHScan is an effective tool for analyzing comparative genomic hybridization data from high-density microarrays. The algorithm is capable of accurately identifying known variable regions and is tolerant of high noise and varying methods of data preprocessing. Statistical analysis is used to define each variable region providing a robust and reliable method for rapid identification of genomic differences independent of annotated gene boundaries.

Algorithms↗
PubMed2005

Evaluation of 3 methods of whole-genome amplification for subsequent metaphase comparative genomic hybridization.

A common aim in cancer research is to investigate mechanisms of malignant progression by genetic analysis of key stages, including pre-malignancy, microinvasion, and micrometastases. As such lesions are small and require microdissection from clinical samples, the amount of DNA that can be recovered is limited and frequently inadequate for commonly used techniques of genomic analysis, such as comparative genomic hybridization (CGH). There is a critical requirement for techniques of whole-genome amplification that minimize representation bias in the amplified sample. Several techniques have been described, although their relative suitability for CGH has not been examined adequately. Here we compare the abilities of degenerate oligonucleotide-primed PCR (DOP-PCR), multiple-strand displacement amplification (MDA), and balanced PCR accurately to amplify limited amounts of template DNA for use in CGH. Amplification by DOP-PCR and MDA, but not balanced PCR faithfully preserved the original genomic content following amplification, as evidenced by generally concordant CGH copy number karyograms. Whereas the amplification products of DOP-PCR were immediately available for labeling and hybridization, the products of MDA required a further digestion step to produce optimal-sized probes for CGH. Moreover, MDA was less reliable overall than DOP-PCR at the lowest starting amount of 10 pg of template DNA. We conclude that DOP-PCR is the method of choice for whole-genome amplification of minute quantities of DNA to enable global genomic analysis to be performed on limited clinical samples.

Metaphase↗