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UCSC Human Genome Browser view of chromosome 2 (http://www.genome.ucsc.edu) with coverage of the optimized clone set (top track), the initial clone set (middle
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Array-based comparative genomic hybridization (aCGH) is a recently developed tool for genome-wide determination of DNA copy number alterations. This technology has tremendous potential for disease-gene discovery in cancer and developmental disorders as well as numerous other applications. However, widespread utilization of a CGH has been limited by...
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... When the values significantly deviate from the baseline, it indicates loss or gain with respect to the reference genome. Initial CNV detection was with arrays having a resolution of~1 Mb (Greshock et al. 2004), and later close to 50 kb . The current generation of genomic arrays contains even millions of probes, and the resolution can be as high as a single exon in genes of interest (Boone et al. 2010). ...
Molecular methods, by which copy number variants (CNVs) detection is available, have been gradually introduced into routine diagnostics over the last 15 years. Despite this, some CNVs continue to be a huge challenge when it comes to clinical interpretation. CNVs are an important source of normal and pathogenic variants, but, in many cases, their impact on human health depends on factors that are not yet known. Therefore, perception of their clinical consequences can change over time, as our knowledge grows. This review summarises guidelines that facilitate correct classification of identified changes and discusses difficulties with the interpretation of rare, small CNVs.
... Similarly, tripartite motif containing 37 (TRIM37 on chr17q23) was a common 5′ partner in three distinct gene fusions with different 3′ partners (Table W2). To further expand our integrative analysis of copy number aberrations and gene fusions, next we used the breast cancer aCGH data[13,14]and observed gene fusion–associated amplicons in MCF7, BT-474, and MDAMB-468, HCC-1187 as seen in our data as well as in an additional panel of cell lines, including ZR-75-30, SUM190, MDA-MB-361, HCC-1428, and HCC-1569 (Figure W2). Clearly, apart from triggering overexpression of constituent genes, our observations strongly suggest that the loci of chromosomal amplifications also serve as " hotspots " for the generation of recurrent gene fusions. ...
... Similarly, tripartite motif containing 37 (TRIM37 on chr17q23) was a common 5′ partner in three distinct gene fusions with different 3′ partners (Table W2). To further expand our integrative analysis of copy number aberrations and gene fusions, next we used the breast cancer aCGH data [13] [14] and observed gene fusion–associated amplicons in MCF7, BT-474, and MDA- MB-468, HCC-1187 as seen in our data as well as in an additional panel of cell lines, including ZR-75-30, SUM190, MDA-MB-361, HCC-1428, and HCC-1569 (Figure W2). Clearly, apart from triggering overexpression of constituent genes, our observations strongly suggest that the loci of chromosomal amplifications also serve as " hotspots " for the generation of recurrent gene fusions. ...
Application of high-throughput transcriptome sequencing has spurred highly sensitive detection and discovery of gene fusions in cancer, but distinguishing potentially oncogenic fusions from random, "passenger" aberrations has proven challenging. Here we examine a distinctive group of gene fusions that involve genes present in the loci of chromosomal amplifications—a class of oncogenic aberrations that are widely prevalent in breast cancers. Integrative analysis of a panel of 14 breast cancer cell lines comparing gene fusions discovered by high-throughput transcriptome sequencing and genome-wide copy number aberrations assessed by array comparative genomic hybridization, led to the identification of 77 gene fusions, of which more than 60% were localized to amplicons including 17q12, 17q23, 20q13, chr8q, and others. Many of these fusions appeared to be recurrent or involved highly expressed oncogenic drivers, frequently fused with multiple different partners, but sometimes displaying loss of functional domains. As illustrative examples of the "amplicon-associated" gene fusions, we examined here a recurrent gene fusion involving the mediator of mammalian target of rapamycin signaling, RPS6KB1 kinase in BT-474, and the therapeutically important receptor tyrosine kinase EGFR in MDA-MB-468 breast cancer cell line. These gene fusions comprise a minor allelic fraction relative to the highly expressed full-length transcripts and encode chimera lacking the kinase domains, which do not impart dependence on the respective cells. Our study suggests that amplicon-associated gene fusions in breast cancer primarily represent a by-product of chromosomal amplifications, which constitutes a subset of passenger aberrations and should be factored accordingly during prioritization of gene fusion candidates.
... Array construction and hybridization protocols have been described previously ). This assay is composed of ~4,100 bacterial artificial chromosome (BAC) clones spaced in ~1 Mb intervals across the genome (Greshock, et al. 2004). Upon construction, a specific effort was made to cover known cancer genes (n = ~400). ...
Identification and characterization of underlying genetic aberrations could facilitate diagnosis and treatment of ovarian cancer. Copy number analysis using array Comparative Genomic Hybridization (aCGH) on 93 primary ovarian tumors identified PI3K/AKT pathway as the most frequently altered cancer related pathway. Furthermore, survival analyses to correlate gene copy number and mutation data with patient outcome showed that copy number gains of PIK3CA, PIK3CB, and PIK3R4 in these tumors were associated with decreased survival. To confirm these findings at the protein level, immunohistochemistry (IHC) for PIK3CA product p110α and p-Akt was performed on tissue microarrays from 522 independent serous ovarian cancers. Overexpression of either of these two proteins was found to be associated with decreased survival. Multivariant analysis from these samples further showed that overexpression of p-AKT and/or p110α is an independent prognostic factor for these tumors. siRNAs targeting altered PI3K/AKT pathway genes inhibited proliferation and induced apoptosis in ovarian cancer cell lines. In addition, the effect of the siRNAs in different cell lines seemed to correlate with the particular genetic alterations that the cell line carries. These results strongly support the utilization of PI3K pathway inhibitors in ovarian cancer. They also suggest identifying the specific component in the PI3K pathway that is genetically altered has the potential to help select the most effective therapy. Both mutation as well as copy number changes can be used as predictive markers for this purpose.
... The correlation of cytogenetic aberrations with disease outcomes has already proven to be reliable and is utilized in (48,74). Diagnostic arrays have been designed for the diagnosis of congenital anomalies, developmental delay, and mental retardation (68,75,76), as well as the detection of chromosomal aberrations in embryos (77)(78)(79). ...
Microarray comparative genomic hybridization (CGH) has proven to be a specific, sensitive, and rapid technique, with considerable advantages compared to other methods used for analysis of DNA copy number changes. Array CGH allows for the mapping of genomic copy number alterations at the sub-microspecific level, thereby directly linking disease phenotypes to gene dosage alterations. The whole human genome can be scanned for deletions and duplications at over 30,000 loci simultaneously by array CGH (~40 kb resolution). Array CGH can be used for analysis of DNA copy number aberrations that cause not only cancer and human genetic disease, but also normal human variation. This review gives the various array CGH platforms and their applications in cancer and human genetics.
... Comparative genomic hybridization was carried out in the '1 Mb BAC' array platform developed at the University of Pennsylvania [14]. DNA probe labelling, aCGH protocol and array data analysis, has been described previously [15]. ...
The search for novel oncogenes is important because they could be the target of future specific anticancer therapies. In the present paper we report the identification of novel amplified genes in lung cancer by means of global gene expression analysis. To screen for amplicons, we aligned the gene expression data according to the position of transcripts in the human genome and searched for clusters of over-expressed genes. We found several clusters with gene over-expression, suggesting an underlying genomic amplification. FISH and microarray analysis for DNA copy number in two clusters, at chromosomes 11q12 and 13q34, confirmed the presence of amplifications spanning about 0.4 and 1 Mb for 11q12 and 13q34, respectively. Amplification at these regions each occurred at a frequency of 3%. Moreover, quantitative RT-PCR of each individual transcript within the amplicons allowed us to verify the increased in gene expression of several genes. The p120ctn and DP1 proteins, encoded by two candidate oncogenes, CTNND1 and TFDP1, at 11q12 and 13q amplicons, respectively, showed very strong immunostaining in lung tumours with gene amplification. We then focused on the 13q34 amplicon and in the TFDP1 candidate oncogene. To further determine the oncogenic properties of DP1, we searched for lung cancer cell lines carrying TFDP1 amplification. Depletion of TFDP1 expression by small interference RNA in a lung cancer cell line (HCC33) with TFDP1 amplification and protein over-expression reduced cell viability by 50%. In conclusion, we report the identification of two novel amplicons, at 13q34 and 11q12, each occurring at a frequency of 3% of non-small cell lung cancers. TFDP1, which encodes the E2F-associated transcription factor DP1 is a candidate oncogene at 13q34. The data discussed in this publication have been deposited in NCBIs Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series Accession No. GSE21168.
... Comparative genomic hybridization was done onto the 1 megabase (Mb) Bacterial Artificial Chromosome (BAC) clone array platform developed at the University of Pennsylvania. In few words, the platform is composed of 4134 BAC clones spaced at 1 Mb intervals, including a direct coverage of approximately 400 known cancer genes [29]. DNA probe labeling, aCGH protocol, and array data analysis have been described else- where [28]. ...
Breast cancer subtypes exhibit different genomic aberration patterns with a tendency for high-level amplifications in distinct chromosomal regions. These genomic aberrations may drive carcinogenesis through the upregulation of proto-oncogenes. We have characterized DNA amplification at the human chromosomal region 13q34 in breast cancer.
A set of 414 familial and sporadic breast cancer cases was studied for amplification at region 13q34 by fluorescence in situ hybridization (FISH) analysis on tissue microarrays. Defining the minimal common region of amplification in those cases with amplification at 13q34 was carried out using an array-based comparative genomic hybridization platform. We performed a quantitative real-time - polymerase chain reaction (qRT-PCR) gene expression analysis of 11 candidate genes located within the minimal common region of amplification. Protein expression levels of two of these genes (TFDP1 and CUL4A) were assessed by immunohistochemical assays on the same tissue microarrays used for FISH studies, and correlated with the expression of a panel of 33 antibodies previously analyzed.
We have found 13q34 amplification in 4.5% of breast cancer samples, but the frequency increased to 8.1% in BRCA1-associated tumors and to 20% in basal-like tumors. Tumors with 13q34 amplification were associated with high grade, estrogen receptor negativity, and expression of EGFR, CCNE, CK5, and P-Cadherin, among other basal cell markers. We have defined a 1.83 megabases minimal common region of genomic amplification and carried out mRNA expression analyses of candidate genes located therein, identifying CUL4A and TFDP1 as the most likely target genes. Moreover, we have confirmed that tumors with 13q34 amplification significantly overexpress CUL4A and TFDP1 proteins. Tumors overexpressing either CUL4A or TFDP1 were associated with tumor proliferation and cell cycle progression markers.
We conclude that 13q34 amplification may be of relevance in tumor progression of basal-like breast cancers by inducing overexpression of CUL4A and TFDP1, which are both important in cell cycle regulation. Alternatively, as these genes were also overexpressed in non-basal-like tumor samples, they could play a wider role in cancer development by inducing tumor proliferation.
... À l'opposé, les clones de grande taille (de 50 à 150 kb) de type BAC (Bacterial Artificial Chromosome) et PAC (Phage Artificial Chromosome) sont des cibles robustes pour la sensibilité de détection du signal d'hybridation, et de plus non limitées aux seuls gènes annotés dans les bases de données génomiques. Les puces couramment développées par les labo- ratoires académiques et certaines firmes commerciales possè- dent en moyenne 3 000 clones de ce type avec une résolution avancée d'une mégabase c'est-à-dire une couverture de 10 % du génome humain, en supposant que l'espacement des cibles est régulier tout au long du génome [8,12,13]. ...
L'identification de gènes responsables d'anomalies du développement passe par des approches de clonage positionnel parmi lesquelles la caractérisation moléculaire d'anomalies chromosomiques. La cartographie précise de réarrangements chromosomiques permet de mieux cerner des gènes candidats impliqués dans les maladies génétiques, grâce à l'utilisation d'outils de cytogénétique et de biologie moléculaire. L'array-CGH a ainsi conduit à identifier des microremaniements chromosomiques jusqu'alors inconnus et à préciser les gènes candidats dans les affections étudiées.Nous présentons dans une première partie, les résultats de la cartographie et de la caractérisation moléculaire de deux anomalies microcytogénétiques associées à un retard mental, une délétion 20q13.33 et une délétion 21q22. Ces données ont contribué à la sélection de gènes candidats et à améliorer la prise en charge clinique et le conseil génétique.Par ailleurs, la caractérisation moléculaire d'une translocation réciproque t(3;18)(p12.3;q23) de novo associée à une dystonie précoce généralisée avec retard de développement a été réalisée. Le gène ROBO1 impliqué dans le guidage axonal s'est révélé interrompu. Des explorations complémentaires ont été conduites (array-CGH, étude de l'ARN, étude protéique, migration cellulaire et séquençage du gène ROBO1 chez d'autres patients avec une dystonie syndromique) afin de mieux cerner les conséquences de cette anomalie sur la survenue de cette dystonie syndromique. Les résultats de ces études soulignent les difficultés d'interprétation liées à la découverte de tels réarrangements et la nécessité d'apporter la preuve de leur implication dans le phénotype observé par des moyens appropriés.
... Array based comparative genomic hybridization (aCGH) was performed onto the platform with ϳ5000 human bacterial artificial chromosomes spaced at an average of 1Mb across the genome developed at the University of Pennsylvania. 25 For hybridization, 1 g of test DNA and 1 g of sex-match pooled normal human DNA (obtained from a set of ten healthy female or male volunteers) were labeled with either Cy3-dCTP or Cy5-dCTP (Amersham Biosciences, UK) incorporated by random priming (Bioprime Labeling Kit, Invitrogen, Carlsbad, CA). Equal amounts of test and reference DNA (1 g each) were mixed and precipitated with Cot1-DNA, 3M NaOAc (pH 7.0) and ethanol. ...
Melanocytic nevi frequently harbor oncogenic BRAF mutations, but only a minority progress to melanoma. In human melanocytes, persistent BRAF(V600E) expression triggers oncogene-induced senescence, which implies that bypass of oncogene-induced senescence is necessary for malignant transformation of melanocytes. We show that a subpopulation of primary human melanocytes with persistent expression of BRAF(V600E) do not enter oncogene-induced senescence, but instead survive despite heightened MAPK activity. Disruption of the p53 pathway using short-hairpin RNA initiated rapid growth of these V600E(+) melanocytes in vitro. The resultant V600E(+)/p53(sh) melanocytes grew anchorage-independently in soft agar, formed pigmented lesions reminiscent of in situ melanoma in artificial skin reconstructs, and were weakly tumorigenic in vivo. Array comparative genomic hybridization analysis demonstrated that the transformed melanocytes acquired a substantial deletion in chromosome 13, which encodes the Rb1 tumor suppressor gene. Gene expression profiling study of nevi and melanomas showed that p53 target genes were differentially expressed in melanomas compared with nevi, suggesting a dysfunctional p53 pathway in melanoma in vivo. In summary, these data demonstrate that a subpopulation of melanocytes possesses the ability to survive BRAF(V600E)-induced senescence, and suggest that p53 inactivation may promote malignant transformation of these cells.
... Melanomas were assayed on a 1-Mb resolution BAC clone-based CGH array designed specifically for cancer analysis (Greshock et al., 2004). One μg of tumor and reference DNA (pooled lymphocyte-derived DNA from 10 individuals) were labeled with Cy3 or Cy5 flourescent dyes respectively (Amersham, Piscataway, NJ) using the BioPrime randomprimed labeling kit (Invitrogen, Carlsbad, CA). ...
A majority of malignant melanomas harbor an oncogenic mutation in either BRAF or NRAS. If BRAF and NRAS transform melanoma cells by a similar mechanism, then additional genetic aberrations would be similar (or random). Alternatively, distinct mutation-associated changes would suggest the existence of unique cooperating requirements for each mutation group. We first analyzed a panel of 52 melanoma cell lines (n = 35, 11, 6 for BRAF*, NRAS*, and BRAF/NRAS(wt/wt), respectively) by array-based comparative genomic hybridization for unique alterations that associate with each mutation subgroup. Subsequently, those DNA copy number changes that correlated with a mutation subgroup were used to predict the mutation status of an independent panel of 43 tumors (n = 17, 13, 13 for BRAF*, NRAS*, and BRAF/NRAS(wt/wt), respectively). BRAF mutant tumors were classified with a high rate of success (74.4%, P = 0.002), whereas NRAS mutants were not significantly distinguished from wild types (26/43, P = 0.12). Copy number gains of 7q32.1-36.3, 5p15.31, 8q21.11, and 8q24.11 were most strongly associated with BRAF* tumors and cell lines, as were losses of 11q24.2-24.3. BRAF* melanomas appear to be associated with a specific profile of DNA copy number aberrations that is distinct from those found in NRAS* and BRAF/NRAS(wt/wt) tumors. These findings suggest that although both BRAF and NRAS appear to function along the same signal transduction pathway, each may have different requirements for cooperating oncogenic events. The genetic loci that make up this profile may harbor therapeutic targets specific for tumors with BRAF mutations.
