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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
•
•
•
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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
Daniel Pinkel & Donna G Albertson (2005) Nature
Genetics, 37:s11-17
• Several genomes can be
compared simultaneously
Daniel Pinkel & Donna G Albertson (2005) Nature
Genetics, 37:s11-17
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)
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
Daniel Pinkel & Donna G Albertson
(2005) Nature Genetics, 37:s11-17
Factors influencing the success of array CGH
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.
2.
3.
4.
5.
6.
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).
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 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
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
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
Findings and implications
•
Widespread DNA copy number alterations in
breast tumors
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•
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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)