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Cytogenetics 101

Douglas Blake, CG(ASCP)CM

Clinical Field Application Scientist

Agilent Technologies


Introduction to aCGH

2 Experimental Considerations

1

3 Experimental Analysis

Agenda


Technology Advancements: Smaller Aberrations

3-5Mb 50-100kb 5-10Mb 0.5Mb >60bp 1bp


Classical cytogenetic analysis


Sample types used in clinical CGH research

Pre-implantation

• Single cell

Pre-natal

• Amniotic Fluid

• Chorionic Villus Sampling

Post-natal

• Blood

• Saliva

Cancer

• Blood

• Bone marrow

• Solid tumor:

• Frozen

• FFPE


How does it work? Typical workflow

Sample Nucleic Acid

Purification Labeling

Hybridization Scanning Data Analysis

Sample QC


CGH Data Interpretation

Not approved for use in diagnostic

procedures

7

Trisomy 21


Chromosome view Gene View

NA14117 46,XY,del(5)(qter>p14:).ishdel(5)

(D5S23-)

Analysis of a Cytogenetic Sample


aCGH Detects Net Copy Changes


What is aCGH


1

3 Experimental analysis

Agenda


Range of Challenges for aCGH


procedures

11

Adapted from Nat Genet. 2005 Jun; 37 Suppl:S11-7


CNV Consideration for Targeted Designs

May 8, 2014

Confidentiality Label

12

• Microarray Content (to include CNV or not)

• Reference DNA (Single vs. Multiple)

• Confirmation (DGV, FISH, Parental, MLPA, etc)

• International Databases/ Consortia


Normal Copy Number Variants (CNVs)

The purpose of your aCGH experiment should dictate inclusion of Normal CNVs in the design

Pros:

• Allows for population/ethnicity studies

• Increase internal database knowledge

• Useful when using a single individual control

Cons:

• Can complicate the analysis

• Requires additional time to cross-reference with external databases

• Can be difficult to interpret if change is


Type of Reference

May 8, 2014


procedures

14

Control Notes Details

Self / Self

• If the same DNA sample is both test and

reference, log ratios should be ‘0’ across the

genome.

• Deviations from ‘0’ indicate false positives

and can be used to predict false positive rates

Any high-quality DNA sample can

be used for a self-self test.

Male / Female

• Here, the expected log2 ratio for the X

chromosome is -1.

• DNA Analytics can calculate additional QC

metrics based on this expectation.

Only normal, non-diseased

samples should be used.

Test DNA /

single individual

“normal” control

•DNA from experiment study is compared to a

known “normal” sample.

•Normal CNVs from control can be eliminated

from analytical consideration

•Allows for SNP incorporation

Genotype reference file required

for known “normal”

Test DNA /

pooled individual

“normal” control

• DNA from a pooled population (n=5-10)

• Many common CNVs can be eliminated from

the evaluation due to a cancelling out effect

Cannot be used in a SNP

evaluation.


Evolution of Array Designs

0 +1 -1

Targeted

WMS

tel

tel

cen

cen

Targeted + 850

BAC or oligo array

0 +1 -1

>3 Mb gaps

Targeted +

1 Mb or 500 kb

0 +1 -1 0 +1 -1

Targeted +

Whole Genome

Chr 7

0.5 - 1 Mb gaps


Methods of Confirmation

Method Notes Details

Parental studies • Array performed on both parents to

determine inheritance status

• Classical cytogenetic studies on parents

• Can be costly for the lab as

reimbursement for this is still

in limbo

FISH studies • FISH to confirm loss/gain identified on array.

• Very inexpensive and quick

• Requires availability of probe

• Small gains (


Public CNV databases

http://www.ncbi.nlm.nih.gov/omim


What is aCGH


1

3 Experimental analysis

Agenda


Scanner output example

2 Constitutional analysis

1

3 Oncology analysis

Experimental Analysis


Scanner output


Analysis Software

Log2 ratio of

intensities to call CNCs

No. of uncut alleles

to call LOH/UPD

Chromosome view Gene view

Aberrations

Customer defined

tracks

Preloaded tracks

Tracks

Genome view


Scanner output example


1

3 Oncology analysis

Agenda


Confirmation of Copy Number Changes trisomy of chr. 21

Amplification

FOUR or more states

Each state corresponds to 0, 1, 2, 3 (or more) copies of the uncut allele

Amplification of chr. 21

SNP

data

CGH

data

BBB

ABB

AAB

AAA


Constitutional Disorders

Deletions


Confirmation of Copy Number Changes e.g. NA09209: hemizygous deletion on chr. 17

SNP

data

CGH

data Deletion at the

beginning of p-arm

Hemizygous deletion

TWO states of alleles appear (only one copy allele):

1) cut copy of the allele (0 copy)

2) uncut copy of the allele (1 copy)

B

A



Duplications



Unbalanced translocation


UPD: uniparental disomy

• Both members of a chromosome pair or segments of a chromosome pair are

inherited from one parent

• UPD can result in an abnormal phenotype when the chromosomes involved are

imprinted, such that only the maternal or paternal allele of the pair is active

Remark: parental

samples needed

to detect

heterodisomy


Detection of UPD complete paternal isodisomy on chr. 15

SNP

data

CGH

data

No heterozygous SNPs

Copy-neutral LOH or UPD

Only TWO states of alleles appear:

1) 0 uncut copies

2) 2 uncut copies

Both copies were

inherited from the same

parent – UPD

BB

AB

AA


Examples of LOH

Detection of Copy Neutral LOH

Confirmation of Deletions and Duplications


Sensitivity to detect mosaicism down to 8%

Valli et al. Molecular Cytogenetics 2011, 4:13

Partial profiles of a sample with a terminal deletion on chromosome 4.

Panel A: Sample’s 100% DNA, B: Synthetic mosaicism at 10% level, C: 8%, D: 7%.

The Agilent arrays are able to detect the deletion at the 8% and 10% level, but not

at the 7% level.



procedures

34

Miller el. al. Amer J. Human Genet 86, 749–764, May 14, 2010 749

Technology Advancements: CGH vs. Karyotype


Review classical analysis


1

3 Oncology analysis

Agenda

2 deletions on p-arm of Chr8

Amplification to 4 copies on

q-arm

SNP shows copy number of 0,

2, 4

AAAA

AABB

BBBB

Breakpoint

AA

AB

BB

CLL Sample

balanced duplication

Amplification to 4 copies on 6p

SNP data show 0, 1, 3, 4 copies; unbalanced duplication

SNPs that were homozygous in the normal show 0 or 4 copies;

SNPs that were heterozygous in the normal now show 1 or 3 copies

AAAA

ABBB

AAAB

BBBB

0

1

2

3 4

4 copies

3 copies

1 copy

0 copy

ALL Sample

unbalanced duplication


SNP data reveals the copy number of the cancer clones

Major clone

Major clone

Minor clone (SNP data is mixture of minor and major clone)

AA AB BB

AAA AAB ABB BBB,

triploid region

AAAAAA AAAAAB ABBBBB BBBBBB

Unbalanced: 1 copy one parent, 5 copy other

parent

AAAAAA AAABBB BBBBBB

Balanced : 3 copies each parent

AML sample: 87% clonal fraction

6

AML Complex Genomic alterations


CGH

3 copies

1 copy

SNP

Triplication

of one allele

Homozygous

deletion

Triplication

of one allele

MDS

Copy Gain LOH

Copy Gain LOH

Copy Loss LOH

0 copy

Heterozygous

deletion

Complex Genomic alterations


P2250_007_2 Chr Cytoband Size (Mb) Start Stop Amp

chr2 p25.3 - p11.2 87 14523 87158839 0.208303

SNP probes Help in calling low-level mosaicism

BBB

ABB

AAB

AAA


AAA

AAB ABB

BBB

Del3p

Dup 5q

AA

AB

BB

A

B

CGH+SNP Array on FFPE Samples


Questions?

Agilent products described are For Research Use Only. Not for

use in diagnostic procedures. User is responsible for US FDA

approval or clearance prior to diagnostic use.

Presentation Title Arial 28pt Bold Agilent Blue · 2016. 9. 4. · S1 CAG EMEAI | Agilent...

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