Transcript Large-scale Copy-Number Variations

Nature Genetics Vol.36 Sept 2004
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Detection of
Large-scale Variation
In the Human Genome
Iafrate, Feuk, Rivera, Listewnik,
Donahoe, Qi, Scherer, Lee
Trevor Pugh
MEDG505 – Genomic Analysis
University of BC
Title Page
Large-scale Copy-Number Variations: 5 W’s & How
What are Large-scale Copy Number Variations?
Who have Large-scale Copy Number Variations?
Where are Large-scale Copy Number Variations?
Why are there LCVs?
When did LCVs arise?
How are LCVs generated?
What are Large-scale Copy-Number Vars.?
@ gains or losses of kb’s-Mb’s of genomic DNA
@ seen in phenotypically normal individuals
> may not be direct cause of genetic disease
> may give rise to disease?
chromosomal rearrangements
subtle phenotypic variation (gene expression)
@ NOT limited to intergenic or intronic regions
Who have Large-scale Copy-Number Vars?
@ applied array CGH to 55 unrelated individuals
Deletion!
Five steps of CGH
@ Isolation of Genomic DNA
from two patients/sources
@ Digestion of Genomic DNA
into short fragments
@ Labeling of each sample with
DIFFERENT detectable dyes
@ SIMULTANEOUS Hybridization
of labeled probes to targets
@ Quantification of amount of
hybridization to each target
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@ spots are large DNA fragments ‘tiled’ ~1MB across
the genome  low resolution, captures 12% of genome
Who have Large-scale Copy-Number Vars?
@ applied array CGH to 55 unrelated individuals
> 39 healthy phenotypes (normal karyotype)
> 16 with known chromosomal abnormalities
... against a pool of (different) normal controls
@ detected *all* known LCVs in 16 abnormal samples
> good sensitivity and specificity
@ on average, detected 12.4 LCVs in normal controls!
> mostly single large insert clones
 up to 2Mb affected!
Who have Large-scale Copy-Number Vars?
@ on average, detected 12.4 LCVs in normal controls
# of LCVs found in:
153
>1 individual
(78)
>10% of individuals
(24)
102
Low frequency
(False Positive rate <1 per 5,264 clones)
@ 26 mapped to previously recognized segmental dupes
@ 13 reside within 100kb gaps of current human genome
sequence presentation
 complicates assembly of final HGP sequence
Where are Large-scale Copy-Number Vars?
RED = copy gains
BLUE = copy losses
Where are Large-scale Copy-Number Vars.?
@ across the genome involving as much as 2MB
@ not limited to intergenic or intronic regions
> can include duplicate genes!
@ confined to localized chromosomal regions
> NOT duplication events of other chromosomes
> tandem copy-number changes
OR
Where are Large-scale Copy-Number Vars.?
@ tandem copy-number changes
> visualized using FISH & fibre-FISH
signals
only from
sister
chromatids
RELATIVE:
RED =
5’ head
GREEN =
3’ tail
Losses
6 duplicates
Normal
9 duplicates
Gains
12 duplicates
> CN gains show more head-to-tail gene copies
Where are Large-scale Copy-Number Vars.?
@ tandem copy-number changes
> verified by QT-PCR
> ratios track FISH & CGH ratios almost perfectly!
When did Large-scale Copy-Number Variations Arise?
@ Unknown!
Why do Large-scale Copy-Number Variations Exist?
@ Unknown!
“Large-Scale Variation Among Human and Great Ape Genomes
Determined by Array Comparative Genomic Hybridization”
Locke, Segraves, Carbone, Archidiacono, Albertson, Pinkel, Eichler
Genome Research Vol 13, Issue 3, 347-357, March 2003
@ gene-rich regions susceptible to CN changes
between humans and great apes
@ local repatterning of hominoid chromosomes in
euchromatic regions
> duplication-driven mechanism of genome evolution
How do LCVs Impact Us?
@ new explanations for individual uniqueness
> lineage studies?
> forensics?
@ increased gene copy number associated with
increased susceptibility to diseases?
> use as genetic markers?
@ added level of complexity to the genome
> comparative genomics?
> phylogenetics?
@ simplification of the genome
> less regulatory networks, more ‘dosage effects’
Summary:
Large-scale Copy-Number Variations (LCVs)
What?
@ gains or losses of kb’s-Mb’s of tandem genomic sequences
Who?
@ varies from person to person, 12.4 LCVs on average
Where?
@ throughout the genome, localized to chromosomal regions
@ not limited to intergenic or intronic regions
When?
@ Unknown! Conjecture: Complex organisms? Plant/Animal?
Why?
@ Unknown!
Conjecture: Gene evolution? Regulatory?
How?
@ new basis for human uniqueness & treatment
@ new ways of interpreting the genome and its interactions
Questions
@ Why do LCVs exist?
When did they arise?
@ Are these LCVs random? If not, what types of genes
would have copy number variation?
@ What possible mechanisms could create LCVs?
@ Is this a genomic simplification or an added level
of complexity?
@ What are the implications of LCVs for current
technologies, databases, and assemblies?
> PCR, gene expression, genetics?
> GenBank, OMIM?
> Human Genome Project?
Questions?
Extra Figures