Biomedical subjects

Richard Segraves

Publications and source records attributed to Richard Segraves.

5 recordsLinked to original sources

PubMed2003

Shaping of tumor and drug-resistant genomes by instability and selection.

Tumors with defects in mismatch repair (MMR) show fewer chromosomal changes by cytogenetic analyses than most solid tumors, suggesting that a greater proportion of the alterations required for malignancy occur in genes with nucleotide sequences susceptible to errors normally corrected by MMR. Here, we used genome-wide microarray comparative genomic hybridization to carry out a higher resolution evaluation of the effect of MMR competence on genomic alterations occurring in 20 cell lines and to determine if characteristic aberrations arise in MMR-proficient and -deficient HCT116 cells undergoing selection for methotrexate resistance. We observed different spectra of aberrations in MMR-proficient compared to -deficient cell lines, as well as among cell lines with different types of MMR-deficiency. We also observed different genetic routes to drug resistance. Resistant MMR-deficient cells most frequently displayed no copy number alterations (16/29 cell pools), whereas all MMR-proficient cells had unique abnormalities involving chromosome 5, including amplicons centered on the target gene, DHFR and/or a neighboring novel locus (7/13 pools). These observations support the concept that tumor genomes are shaped by selection for alterations that promote survival and growth advantage, as well as by the particular dysfunctions in genes responsible for maintenance of genetic integrity.

Base Pair Mismatch↗

PubMed2003

Large-scale variation among human and great ape genomes determined by array comparative genomic hybridization.

Large-scale genomic rearrangements are a major force of evolutionary change and the ascertainment of such events between the human and great ape genomes is fundamental to a complete understanding of the genetic history and evolution of our species. Here, we present the results of an evolutionary analysis utilizing array comparative genomic hybridization (array CGH), measuring copy-number gains and losses among these species. Using an array of 2460 human bacterial artificial chromosomes (BACs) (12% of the genome), we identified a total of 63 sites of putative DNA copy-number variation between humans and the great apes (chimpanzee, bonobo, gorilla, and orangutan). Detailed molecular characterization of a subset of these sites confirmed rearrangements ranging from 40 to at least 175 kb in size. Surprisingly, the majority of variant sites differentiating great ape and human genomes were found within interstitial euchromatin. These data suggest that such large-scale events are not restricted solely to subtelomeric or pericentromeric regions, but also occur within genic regions. In addition, 5/9 of the verified variant sites localized to areas of intrachromosomal segmental duplication within the human genome. On the basis of the frequency of duplication in humans, this represents a 14-fold positional bias. In contrast to previous cytogenetic and comparative mapping studies, these results indicate extensive local repatterning of hominoid chromosomes in euchromatic regions through a duplication-driven mechanism of genome evolution.

Animals↗

PubMed2003

Whole genome analysis of genetic alterations in small DNA samples using hyperbranched strand displacement amplification and array-CGH.

Structural genetic alterations in cancer often involve gene loss or gene amplification. With the advent of microarray approaches for the analysis of the genome, as exemplified by array-CGH (Comparative Genomic Hybridization), scanning for gene-dosage alterations is limited only by issues of DNA microarray density. However, samples of interest to the pathologist often comprise small clusters of just a few hundred cells, which do not provide sufficient DNA for array-CGH analysis. We sought to develop a simple method that would permit amplification of the whole genome without the use of thermocycling or ligation of DNA adaptors, because such a method would lend itself to the automated processing of a large number of tissue samples. We describe a method that permits the isothermal amplification of genomic DNA with high fidelity and limited sequence representation bias. The method is based on strand displacement reactions that propagate by a hyperbranching mechanism, and generate hundreds, or even thousands, of copies of the genome in a few hours. Using whole genome isothermal amplification, in combination with comparative genomic hybridization on cDNA microarrays, we demonstrate the ability to detect gene losses in yeast and gene dosage imbalances in human breast tumor cell lines. Although sequence representation bias in the amplified DNA presents potential problems for CGH analysis, these problems have been overcome by using amplified DNA in both control and tester samples. Gene-dosage alterations of threefold or more can be observed with high reproducibility with as few as 1000 cells of starting material.

Breast Neoplasms↗

PubMed2002

Transformation of follicular lymphoma to diffuse large cell lymphoma is associated with a heterogeneous set of DNA copy number and gene expression alterations.

Genomic aberrations in a series of paired biopsy samples from patients who presented initially with follicle center lymphoma (FCL) and subsequently transformed to diffuse large B-cell lymphoma (DLBCL) were measured by array comparative genomic hybridization (CGH). The consequences of these aberrations on gene expression were determined by comparison with expression analysis on these specimens using cDNA microarrays. A heterogeneous pattern of acquired genomic abnormalities was observed upon transformation, some of which were recurrent in small subsets of patients. Some of the genomic aberration acquired upon transformation, such as gain/amplification of 1q21-q24, 2p16 (REL/BCL11A gene loci), 3q27-q29 (including the BCL6 locus), 7q11.2-q22.1, 12pter-q12, 18q21 (including the BCL2 locus) and Xq, and deletion of 6q22-q24, 13q14-q21 and 17p13 (P53 locus) have been previously implicated in the FCL/DLBCL pathogenesis. In addition, novel genomic imbalances not previously reported in association with FCL transformation, such as overrepresentation of 4p12-pter, 5p12-p15, 6p12.3-p21, 9p23, 9q13-q31, 16q, 17q21, and loss of 1p36.3, 4q21-q23, 5q21-q23, 9q31-qter, 11q24-q25, and 15q23, were identified. We observed a differential expression profile of many genes within regions of gain and deletion upon transformation, including novel target genes associated with FCL transformation. However, other genes did not show deregulated expression despite their location within these areas. In summary, the combination of array CGH and expression analysis provides a more comprehensive picture of the transformation of FCL to DLBCL. This process is associated with the acquisition of a variable spectrum of genomic imbalances affecting recurrent chromosomal areas that harbor overexpressed or underexpressed genes targeted upon transformation.

Allelic Imbalance↗

PubMed2002

Fully automatic quantification of microarray image data.

DNA microarrays are now widely used to measure expression levels and DNA copy number in biological samples. Ratios of relative abundance of nucleic acids are derived from images of regular arrays of spots containing target genetic material to which fluorescently labeled samples are hybridized. Whereas there are a number of methods in use for the quantification of images, many of the software systems in wide use either encourage or require extensive human interaction at the level of individual spots on arrays. We present a fully automatic system for microarray image quantification. The system automatically locates both subarray grids and individual spots, requiring no user identification of any image coordinates. Ratios are computed based on explicit segmentation of each spot. On a typical image of 6000 spots, the entire process takes less than 20 sec. We present a quantitative assessment of performance on multiple replicates of genome-wide array-based comparative genomic hybridization experiments. By explicitly identifying the pixels in each spot, the system yields more accurate estimates of ratios than systems assuming spot circularity. The software, called, runs on Windows platforms and is available free of charge for academic use.

Breast Neoplasms↗