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Comparative Genomic Hybridization (CGH)

Comparative Genomic Hybridization (CGH). Outline. Introduction to gene copy numbers and CGH technology DNA copy number alterations in breast cancer (Pollack et.al., PNAS (2002)) Copy number polymorphism in human genomes (Sebat et.al ., Science (2004)).

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Comparative Genomic Hybridization (CGH)

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  1. Comparative Genomic Hybridization(CGH)

  2. Outline • Introduction to gene copy numbers and CGH technology • DNA copy number alterations in breast cancer (Pollack et.al., PNAS (2002)) • Copy number polymorphism in human genomes (Sebat et.al., Science (2004))

  3. Alteration in DNA Copy Number: amplification and deletion • Abnormal quantity of appearance of a genomic region in the genome. • Size: single gene - whole chromosome • Abnormality: deletion – amplification • Some variations among normal individuals • Can cause defects in human development • Contributors to cancer • Can effect function and gene expression

  4. Alteration in DNA Copy Number: possible mechanism Molecular Biology of the Cell, Alberts et. al. (4th eddition, figure 23-33) Molecular Biology of the Cell, Alberts et. al. (4th eddition, figure 23-28)

  5. Array Based Comparative Genomic Hybridization • Goal: to detect copy number alterations using a gene chip • Ideally, the signal intensity is proportional to copy number • Several genomes can be compared simultaneously Daniel Pinkel & Donna G Albertson (2005) Nature Genetics, 37:s11-17 Daniel Pinkel & Donna G Albertson (2005) Nature Genetics, 37:s11-17

  6. Technical consideration in array CGH Hybridization signals • Affected by base composition, repetitive sequences, chosen probes, saturation of the array, double-strand association etc. • Lower signals obtained for lower complexity probes (cDNA and PCR products) Genome characteristics • Hybridization of repetitive elements, should be blocked Copy number measurements • Difficult to detect deletions • Low-copy reiterated sequences • Copy-number polymorphism • Heterogeneous specimens (cells with altered DNA mixed with normal cells)

  7. Technical consideration in array CGH Specimen preparation • Differences in quality of cell lines, frozen/fresh/fixed tissue • Heterogeneous specimens • Extraction of DNA Data analysis • Significance of signal ratios Factors influencing the success of array CGH Daniel Pinkel & Donna G Albertson (2005) Nature Genetics, 37:s11-17

  8. Applications of array CGH in oncology • Use in clinical trials for CLL drugs (to determine relationship between therapeutic options and genomic aberrations) • Association of DNA copy-number with prognosis in a variety of tumors (prostate, breast, gastric, lymphoma) • Detecting a region and not a gene • Not always found in correlation with gene expression • Wide range of genomic phenotypes • Ongoing genomic instability results in heterogeneity

  9. Microarray analysis reveals a major direct role of DNA copy number alteration in the transcriptional program of human breast tumors(Pollack et. al. (2002) PNAS 99:12963-8) • Analysis of DNA copy number in breast tumors • Array based CGH • High resolution (gene-by-gene) mapping of boundaries • Parallel microarray measurements of mRNA level Daniel Pinkel & Donna G Albertson (2005) Nature Genetics, 37:s11-17

  10. Materials Tumors and cell lines • 44 breast tumors • Infiltrating ductal carcinoma • Intermediate-grade • >50% tumor cells • 10 breast cancer cell lines DNA labeling and hybridization • 6,691 cDNA genes on array (Genomic locations from UCSC) • Reference DNA was taken from normal female leukocyte

  11. Estimating significance of altered fluorescence ratios • 5-nearset neighbors average smoothing • For normal data: for each gene i find window size k(i) giving highest positive and negative average - val0(i). • Find cut points Cup and Clow so that overall proportion of false positives is α/2 in each tail of distribution. • For tumor data: for each gene i find window size k(i) giving highest positive and negative average - val(i). • Mark as significant all values > Cup or < Clow. • FDR rate is nα/s (for each sample α was chosen so that FDR was closest to 0.01).

  12. Performance of analysis • Mean moving average ratios of autosomal and X-chromosomal cDNA from samples with variation in chromosome X number • 227 X-chromosomal cDNA • Gains and losses identifiable

  13. Numerous DNA copy number alterations • Changes in each sample and on each chromosome • Magnitude lower in tumors • Several gains and losses common to most samples (consistent with published studies) • Number of alterations significantly higher in high-grade, estrogen receptor negative and TP53 mutant tumors.

  14. Variation in copy number mapping to chr 17 ERBB2 (HER2) oncogene GRB7 MLN64

  15. Parallel microarray measurements of mRNA level Goal: • Highly amplified genes that are highly expressed are strong candidate oncogene • Global impact of widespread DNA copy number alterations on gene expression in tumor cells • mRNA levels were measured for a subset of samples and genes • 4 cell line, 37 tumors • 6,095 genes

  16. Parallel microarray measurements of mRNA level • 117 high level DNA amplifications (91 different genes) • 62% (54 genes) found associated with at least moderately elevated mRNA • 12/54 genes are oncogenes or candidates • 42% (36 genes) found associated with highly elevated mRNA.

  17. Influence of DNA copy-number on mRNA levels • Divided genes to five classes representing: DNA deletion No change Low level amplification Medium level amplification High level amplification • Significant correlation between mRNA level and copy number across groups • On average a 2 fold change in copy number was accompanied by 1.4 and 1.5 fold changes in mRNA level

  18. Influence of DNA copy-number on mRNA levels • Distribution of 6,095 correlations between copy number and expression levels • Significant right shift • Reflects global influence of DNA copy number alterations on gene expression

  19. Influence of DNA copy-number on mRNA levels Percent of variance in gene expression (tumors) explained by variation in gene copy number • At least 7% of observed variation in mRNA levels can be explained by variation in copy number

  20. Findings and implications • Widespread DNA copy number alterations in breast tumors • Deletion of TSG and amplification of oncogenes • Many other alterations, may cause an imbalance in expression  imbalance in physiology and metabolism  further chromosomal instability  tumorigenesis • High degree of copy number-dependent gene expression • 62% of highly amplified genes demonstrate elevated expression levels • Elevation in expression of an amplified gene cannot alone indicate a candidate oncogene

  21. Large-Scale Copy Number Polymorphism in the Human Genome(Sebat et. al. (2004) Science 305:525-8) • Array based CGH of 20 individuals • Array with 85,000 probes representing human genome (Bgl II, Hind III) • Found 76 unique germ line CNPs (Copy Number Polymorphism) • 11/12 CNPs validated by FISH and other methods • No CNPs observed on chrX • Enrichment of segmental duplications in CNPs • Some CNPs involve genes related to neurological disease, cancer and obesity

  22. Large-Scale Copy Number Polymorphism in the Human Genome(Sebat et. al. (2004) Science 305:525-8)

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