Molecular Cytogenetics tools

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Molecular Cytogenetics tools
Joris Vermeesch
October 4, 2008 Postgraduate course in human
genetics
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Conventional Cytogenetics
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SYNCHRONISATION OF THE CELL CULTURES Metotrexate/T
hymidine
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HYBRIDISATION
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PROBES

• Gekloneerde DNA fragmenten
• Plasmid ( 10 kb)
• Cosmid ( 40 kb)
• BAC/PAC/P1 (150 kb)
• YACs ( 1000 kb)
• Volledige chromosomen resulteren in painting
  probes
• (Flow sorting/microdissectie/somatische cell
  hybriden)
• Genomisch DNA
• Oligonucleotides

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LABELING OF THE PROBES
Nick translatie
Random prime labeling
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DETECTION
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METAFASE FISH(resolutie 3-5Mb)
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INTERFASE FISH(resolutie 500kb-2 Mb)
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FIBER FISH(resolutie 10 kb-200 kb)
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SNP DETECTIE(nucleotide resolutie)
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DIFFERENT COLOUR FISH(Metafasen)
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DIFFERENT COLOUR FISH(Interfasen)
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MULTI-COLOUR FISH (MFISH)
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MULTI-COLOUR FISH (MFISH)
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LABELING SCHEMA
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Spectral karyotyping
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Flow sorting chromosomen
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SOMATISCHE CELL HYBRIDEN
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CHROMOSOOM MICRODISSECTIE
Amplify
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MULTIKLEUREN-BANDING FISH
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CONFOCAL MICROSCOPY
CLSM of LSCM is used to obtain 3-D images. The
most important feature is to obtain sharp images
from relatively thick specimen. By using
laserligth light is projects as points. By using
spatial filters out-of-focus light is eliminated.

http//micro.magnet.fsu.edu/primer/virtual/confoca
l/index.html
http//www.olympusfluoview.com/theory/
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DECONVOLUTION MICROSCOPY
By mathematical filtering scattering light is
removed from the foacal plane. This results in
an images with less noise, a sharper image and a
better 3 D resolution.
www.olympusmicro.com/primer/virtual/confocal/index
.html
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3D reconstructie van deconvulutie microscopie
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REAL TIME CONFOCAL MICROSCOPY
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Comparative Genome Hybridisation (CGH)
Chromosomes on glass slides
deletion
control DNA
Wash
Hybridization
patient DNA
duplication
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Molecular karyotyping
Array of DNA fragments
Duplication
Deletion
Patient DNA
Control DNA
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Array targets

• BACs
• Cosmids, fosmids
• PCR products
• cDNAs
• oligonucleotides

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Array production
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Array protocol FISH

• Probe labeling
• Prehybridisation
• Hybridisation
• Washing
• Drying

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Array scanning
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DETECTION OF DELETIONS
46,XX,del(10)(q25.1q26.11)de novo
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DETECTION OF DUPLICATIONS
No diagnosis
46,XY,der(22),ins(2216)(p13p13.2p13.3)de novo
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DETECTION OF MOSAICS
Bernard Thienpont
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• Rapid identification of chromosomal breakpoints

Amplify
Liesbeth Bacx Hilde Van Esch
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• Affymetrix GeneChip Human Mapping SNP Array Set
  for genotype-based applications and
  high-resolution copy number variation detection

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From probe intensities to genotype calls and CNV
detection
Single hybridisations
G
For CNV detection compare SNP-probe intensities
to a reference pool.
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Child
Father
Mother
CME 80286
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Single cell molecular karyotyping
Amplify
Cédric Le Caignec
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250K SNP-array and single cell analyses
Xq terminal deletion (58 Mb)
18p terminal duplication (14 Mb)
Posterior P
BAC-aCGH
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MOLECULAR KARYOTYPING

• TECHNICAL ASPECTS

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Technical aspects
Is this a normal or an abnormal molecular
karyotype?
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Answer depends on premises

• Technical premises
• Quality I Thresholding
• Quality II Segmentation algoritms
• Qulaility III Dynamic range
• Biological premises
• Polymorphisms?
• Benign or malignant copy number variation

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Fixed tresholds
Vissers et al., 2003
Ishkanian et al., 2004
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Fixed tresholds
0.5
0.3
-0.3
-0.5
0.5
0.3
-0.3
-0.5
Problem number of false positives dependens on
variation of intensity ratios
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results part II data analysis - some
statistics

• 2 x stdev ? 2.5 1/40
• 3 x stdev ? 0.14 1/720
• 4 x stdev ? 0.003 1/30 000

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results part II data analysis - some
statistics
µdel
-0,75
4 x stdev
stdev
stdev
stdev
lt 0.1
lt 0.1
lt 0.1
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results part II data analysis - some
statistics
µdel
-0,75
4 x stdev
stdev
stdev
stdev
lt 0.1
lt 0.1
lt 0.1
-1
-0,8
-0,6
-0,4
-0,2
0
0,2
0,4
0,6
0,8
1
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Tresholding floating threshold
47,XX,13/46,XY
0.58
0.58-2SD
4SD
0
-4SD
Chromosomes 12 13 14
4SDlog2(3/2)-2SD or SD0.096
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tresholding Avoid false positives/false
negatives

• Segmentation algoritms
• Hidden Markov
• CNAT,CNAG
• .

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Quality II segmentation
CME 80286
Child
Father
Mother
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Quality III Dynamic range
Friedman et al., AJHG, 2007
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Array quality Dynamic range

• Factors influencing dynamic range
• BAC amplification quality
• Hybridisation conditions
• CotI quality

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MOLECULAR KARYOTYPING

• CLINICAL ASPECTS

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Very high incidence of submicroscopic imbalances
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Apparently balanced translocations the majority
is unbalanced!!!

• 59 cases
• 41 apparently balanced translocations
• 27 patients 40 (11/27) unbalanced
• 22 (6/27) with deletions at the translocation
  breakpoints
• 18 (5/27) with complex rearrangements
• 14 fetuses all normal
• 18 complex rearrangements 16/18 (89) unbalanced
• 13 patients
• 3 fetuses
• 2 females with repeated abbortions

De Gregori et al., J. Med. Gen. 2007
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From diagnosis to prognosis

• Patient
• Pulmonary valve stenosis
• Cleft uvula
• Mild dysmorphism
• Mild learning difficulties
• High myopia

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From diagnosis to prognosis
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Screening 900 patients with MR/MCA 167 (20)
imbalanced (2007)

• Outcome
• deletions 106
• duplications 43
• mosaicisms 3
• unbalanced translocation 13
• Sizes of anomalies
• 1 clone 1/3 (100kb-3 Mb)
• 2 to 5 clones 1/3 (2 6 Mb)
• gt 5 clones 1/3 (gt 6 Mb)
• Origin of the anomaly
• De novo 3/4
• Familial 1/4

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The imbalances are scattered across the genome
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Conclusion

• Array CGH should be part of the diagnostic
  work-up of patients with MCA/MR

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What causes us (humans) to differ from one
another?
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Large scale copy number variation !!
Estimated to be 12 of genome 400 Mb
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CONCLUSION

• WE ARE ALL ABNORMAL!!

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The challenge Which imbalances are causal for
the phenotype?
Conventional wisdom
Recurrent imbalances with same phenotype are
causal
The larger the size, the more likely causal
Population embedded CNVs are benign
Inherited imbalances are benign while de novo
imbalances are causal
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Identifying recurrent imbalances and phenotypes
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The challenge Which imbalances are causal for
the phenotype?
Conventional wisdom
Recurrent imbalances with same phenotype are
causal
The larger the size, the more likely causal
Population embedded CNVs are benign
Inherited imbalances are benign while de novo
imbalances are causal
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The challenge Which imbalances are causal for
the phenotype?
Malignant imbalances
Benign copy number variation
1 bp
10 Mb
Deletion or duplication size
Size alone is not a good determinant!
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The challenge Which imbalances are causal for
the phenotype?
Conventional wisdom
Recurrent imbalances with same phenotype are
causal
The larger the size, the more likely causal
Population embedded CNVs are benign
Inherited imbalances are benign while de novo
imbalances are causal
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Genome variation Database Map all benign
variation

• Database of genomic variants May 2008
• Redon et al. Nature, 2008

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The challenge Which imbalances are causal for
the phenotype?
Conventional wisdom
Recurrent imbalances with same phenotype are
causal
The larger the size, the more likely causal
Population embedded CNVs are benign
Inherited imbalances are benign while de novo
imbalances are causal
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Mendelian CNVs a paradigm shift in genetics

• Inherited apparently benign CNVs CAN cause
  disease

Mendelian CNVs is the term coined here to
indicate benign CNVs which can cause disease
dependent on either copy number state,
inheritance pattern or genetic and environmental
background.