Comparative Genomic Hybridization (CGH). Outline Introduction to gene copy numbers and CGH...
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Transcript of Comparative Genomic Hybridization (CGH). Outline Introduction to gene copy numbers and 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))
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
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)
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
Technical consideration in array CGHHybridization 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)
Technical consideration in array CGHSpecimen 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
Dan
iel P
inke
l & D
onna
G A
lber
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(2
005)
Nat
ure
Gen
etic
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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
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
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
Estimating significance of altered fluorescence ratios
1. 5-nearset neighbors average smoothing
2. For normal data: for each gene i find window size k(i) giving highest positive and negative average - val0(i).
3. Find cut points Cup and Clow so that overall proportion of false positives is α/2 in each tail of distribution.
4. For tumor data: for each gene i find window size k(i) giving highest positive and negative average - val(i).
5. Mark as significant all values > Cup or < Clow.
6. FDR rate is nα/s (for each sample α was chosen so that FDR was closest to 0.01).
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
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.
Variation in copy
number mapping to
chr 17
ERBB2 (HER2) oncogene
GRB7
MLN64
Parallel microarray measurements of mRNA levelGoal:
• 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
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.
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
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
Influence of DNA copy-number on mRNA levels
• At least 7% of observed variation in mRNA levels can be explained by variation in copy number
Percent of variance in gene expression (tumors) explained by variation in gene copy number
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
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
Large-Scale Copy Number Polymorphism in the Human Genome
(Sebat et. al. (2004) Science 305:525-8)