Comparative Genomic Hybridization (CGH)

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Comparative Genomic Hybridization(CGH)
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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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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

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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)
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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, 37s11-17
Daniel Pinkel Donna G Albertson (2005) Nature
Genetics, 37s11-17
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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)

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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, 37s11-17
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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

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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 9912963-8)

• 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, 37s11-17
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Materials

• Tumors and cell lines
• 44 breast tumors
• Infiltrating ductal carcinoma
• Intermediate-grade
• gt50 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

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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 a/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 gt Cup or lt Clow.
6. FDR rate is na/s (for each sample a was chosen so
   that FDR was closest to 0.01).

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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

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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.

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

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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.

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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

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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

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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

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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

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Large-Scale Copy Number Polymorphism in the Human
Genome(Sebat et. al. (2004) Science 305525-8)

• 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

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Large-Scale Copy Number Polymorphism in the Human
Genome(Sebat et. al. (2004) Science 305525-8)