Download Chromosomal Microarray Analysis

Chromosomal Microarray
Analysis
(CMA)
Medical Genetics Laboratories
Department of Molecular and Human Genetics
Baylor College of Medicine
Table of Contents
•
•
•
•
•
•
•
•
•
•
•
Overview of CMA
Examples of Common Findings
Examples of Mosaicism
Examples of Complex Abnormalities
Examples of Small Copy Number Variants
CMA Comprehensive-CMA plus SNPS
Resolving Variants of Uncertain Significance
Prenatal CMA Considerations
Prenatal CMA Case Examples
Types of Cancer Arrays
Conclusion
Overview of CMA
Chromosomal Microarray Analysis
• CMA is an array-based comparative
genomic hybridization methodology that
allows for analysis of the chromosomes for
a large number of genetic disorders.
• With a single test, CMA can identify the
abnormalities that are detectable by both
routine chromosome analysis and FISH
analysis.
• CMA has greater sensitivity than older
methods of chromosome analysis.
Genomic Resolution
Karyotype [4-5 Mb, whole genome]
FISH [40 to 250 kb per probe, single site]
CMA [average resolution ~30kb, whole genome]
Limits of Resolution
Chromosome vs. Array CGH Analysis
Chromosome 1
Limits of detection for G-banded
chromosome analysis is 4-5 Mb
4 Mb region of microarray data
showing a 1 Mb deletion
encompassing ~70 oligos
What is included?
• Whole genome copy number analysis
• Coverage is more dense at genomic sites associated with
known genetic conditions
– Currently 180,000 to 400,000 probes (oligos) covering the
genome (depending on version)
– Exon coverage of 1700-4400 disease-associated genes
(depending on version)
• Pericentromeric regions
– useful for detecting marker chromosomes
• Subtelomeric regions
• Backbone coverage (30kb resolution)
• Verification of results by FISH analysis and/or partial
karyotype when indicated
• Parental studies to determine if observed copy number
changes are inherited or de novo
CMA Process
[A] Experimental Procedure
Patient
[B] Laser Scanner
[D] Array Profile
Control
Mix
Hybridization of
genomic DNA to
the array of small
DNA fragments
(oligo probes)
[C] Actual Array
Laser Scanner
Duplication
Deletion
Shinawi, M. and Cheung, S.W. (2008) The array CGH and its clinical applications. Drug discovery today, 13, 760-70.
1. Evolution of CMA - increasing pixels!
VERSION
Feb 2004
July 2005
Nov 2006
Mar 2007
Feb 2008
June 2009
V4 BAC
V5 BAC
V6 BAC
V6 OLIGO
V7 OLIGO
V8 OLIGO
366 BACs
853 BACs
1475 BACs
44K oligos
105K oligos
180K
oligos
Genomic disorders
40
75
>140
>140
420 genes
(+61 regions *)
>1700 genes
(exons)
Subtelomeric regions
41
41
41
41
41
41
# of clones / subtel
~4
~10
~10
~10 (x20)
NA
NA
~ 5 Mb
~ 10 Mb
~ 10 Mb
~ 10 Mb
NA
NA
none
43
43
43
43
43
~3
~ 3-5
~ 3-5
~3-5 (x20)
NA
NA
NA
1 clone per
1 band
1 clone per
1 band
30 kb
30 kb
290
290
More
Disorders
mito
+ exon
coverage
TBD
Interrogating probes
Coverage per subtel
Pericentromeric regions
# clone / region
Backbone coverage
NA
LCR regions
Design
Improvement
Detection Rate
FISH test
+ subtel
More
Disorders
+ subtel
+ pericentr
More
disorders
+ each chr
band
BAC clones
 oligo
emulation
More
Disorders
+ mito
+ LCR regions
6.50%
9.04%
~12%
~12.5%
~15.4%
Evolution of CMA - increasing pixels!
Whole Genome Coverage
N >46,000
June 2009
Oct 2010
July 2011
July 2011
2012
V8.0 OLIGO
+SNP Screen
V 8.1.1 OLIGO
V 8.3 OLIGO
+SNP Screen
V9.0 OLIGO
+SNP Screen
VERSION
V8 OLIGO
Interrogating
probes
180K Oligos
400K Oligos
180K Oligos
400K Oligos
400K Oligos
>1700 genes
Exon coverage
HG18
>1700 genes
Exon coverage
HG18
1779 genes
Exon coverage
HG19
1936 genes
Exon coverage
HG19
4864 genes
Exon coverage
HG19
Subtelomeric
regions
41
41
41
41
41
Pericentromeric
regions
43
43
43
43
43
30 kb
30 kb
30 kb
30 kb
30 kb
290
290
290
290
290
More Disorders
Mito
+ exon
coverage
More Disorders
Mito
+ exon coverage
+
120K SNP
More Disorders
Mito
+ exon coverage
Additional 79
genes
Genomic disorders
Backbone
coverage
LCR regions
Design
Improvement
More Disorders
More Disorders
Mito
Mito
+ exon coverage
+ exon coverage +
Additional 200
Additional 2500
genes +120K SNP genes +120K SNP
38 non-coding
regulatory
elements
Advantages of CMA
• Screens for a large number of disorders
simultaneously
• Detects conditions that are difficult to identify
clinically
– Atypical or mild phenotypes; e.g., VCFS/DiGeorge
– Conditions that lack distinctive features
• Detects deletions and duplications simultaneously
• Detects submicroscopic unbalanced chromosome
rearrangements
• Detects mosaicism (as low as 10%)
Cheung et al. (2007) Am J Med Genet A. (15):1679-86
• Detects interstitial subtelomeric deletions/
duplications
Limitations of CMA
• Does not detect balanced translocations,
inversions, low level mosaicism, point mutations
• Can detect copy number variants (CNVs) of
unknown clinical significance
– Most are easily resolved by parental studies, however,
clinicians should carefully evaluate parental phenotypes
and developmental histories so data can be
appropriately interpreted.
– CNVs <500 kb containing no identified genes at time of
analysis are not reported
– CNVs >300 kb containing genes, even if clinical
significance is unknown, are reported
– CNVs <300 kb must contain a gene known to be
associated with disease in order to be reported
Examples of
Common Findings
Normal Result
Trisomy 21
Chromosome 21-specific plot
DiGeorge Syndrome / VCFS
Top: Whole genome view of CMA data
Bottom:
• Left: FISH confirmation
• Inset: Partial karyotype of chromosome 22
(arrow points to deleted 22)
• Right: Chromosome 22-specific oligo plot
of CMA data.
DiGeorge Syndrome / VCFS
del 22q11.2
Microduplication 22q11.2 Syndrome (3 Mb )
• Duplication of the DiGeorge /
Velocardiofacial Syndrome region on
chromosome 22q11.2
Microduplication 22q11.2 Syndrome (3 Mb )
Chromosome 22
Partial karyotype of chromosome 22
(arrow indicates duplicated 22)
FISH confirmation
3 red signals indicates duplication
Example of Mosaicism
Mosaicism Example
Indication – Microcephaly, congenital vertical talus
arr 18q21.2q23(47898780-76103255)x1.nuc ish 18q21.2(RP1125O3x1)[45/200]dn
Chromosomal Microarray Analysis revealed an approximately 28.2 Mb LOSS in
copy number in the distal and subtelomeric regions of the long arm of
chromosome 18 suggestive of mosaicism. This deletion includes the critical
region of chromosome 18q deletion syndrome (OMIM 601808). FISH analysis and
partial chromosome analysis revealed mosaicism for a deletion in the long arm of
one chromosome 18 in 22% (45/200) of interphase cells examined. The remaining
78% (155/200) of cells showed a normal hybridization pattern.
UPDATE: Parental FISH analysis with the above clone showed no evidence of the
same LOSS in the father (KCL144199).
UPDATE: Parental FISH analysis with the above clone showed no evidence of the
same LOSS in the mother (KCL 146866). Therefore, this result most likely
represents a de novo event. Genetic counseling is warranted.
Mosaicism (contin’d)
Indication – Microcephaly, congenital vertical talus
Examples of
Complex Abnormalities
Complex X Chromosome Abnormality
Complex X Chromosome Abnormality
Chromosome X-specific plot
Complex Chromosome 1 Abnormality
A Chr 1
BCM array
B
Nimblegen
2.1
1q41q42 microdeletion
syndrome region
not duplicated
TAR region
duplication
van der Woude
C
D
E
RP11-279E18
D1Z1
RP11-339I11
D1Z1
4.7 Mb (del)
1q32.2
3.1 Mb
(dup)
4.4 Mb
(dup)
1q42.12
1q42.3q43
221,378,468
224,471,629
232,592,278
236,988,147
0.3 Mb
nml
1.8 Mb (del)
205,060,076
209,804,762
AKT3
not deleted
F
G
RP11-478H16
RP5-1090A23
del 1q43
237,337,843
239,105,025
RP11-478H16
RP5-1090A23
Complex Chromosome 1 Abnormality
A. CMA identified a complex rearrangement including two gains and
two losses in chromosome 1q.
B. High density array CGH using Nimblegen 2.1M array showed a 0.3
Mb single copy sequence between the distal duplication and deletion.
C. Chromosome analysis showed that the gained materials (red arrow)
due to the duplications were translocated into the 1q32 region.
D-G. FISH analyses confirmed the deletions in 1q32.2 (D) and 1q43 (E)
and the two copy number gains (F). In addition, FISH analysis on
metaphase cells (G) suggested an inversion between the two regions
with copy number gains and showed the duplicated segments of
1q42.12 and 1q42.3q43 were located next to each other as indicated by
a white arrow.
Liu et al. Cell In Press
Evaluation for a Marker chromosome
Evaluation for a Marker Chromosome
Chromosome 1-specific plot
marker
CMA identified that the marker
chromosome originated from
chromosome 1.
Examples of Small
Copy Number Variants
(including exon deletions)
Deletion of an Exon of ERBB4 on 2q34
INTERPRETATION OF RESULTS:
Chromosomal Microarray Analysis revealed a LOSS in copy number in the
distal long arm of chromosome 2, spanning a minimum of 0.229 Mb and a
maximum of 0.296 Mb. This deletion disrupts the ERBB4 (erythroblastic
leukemia viral oncogene homolog 4) gene. A recent publication describes
haploinsufficiency of ERBB4 gene in a patient with early myoclonic
encephalopathy and profound psychomotor delay (Eur J Hum Genetics. 2009
Mar 17(3):378-82). Clinical correlation is recommended and genetic
counseling is warranted.
Deletion of an Exon of ERBB4 on 2q34
Whole chromosome 2
Deletion of Exons of EP300 on 22q13.32
• Figure 3 A. Profile of the microarray analysis showing
the deleted region as indicated in the red circle [the gain
on Xp as shown in green dots is also present in the
mother (data not shown)];
• B. The deleted oligos displayed in the UCSC genome
browser corresponds to the exons of the CREBBP gene.
• C. The MLPA profile demonstrated copy number
changes in the 2 exons of the CREBBP gene (exons 2728).
• D. The deletion profile is present in the child but not in
the parents indicating the deletion is de novo in origin.
Deletion of Exons of EP300 on 22q13.32
A
B
C
CREBBP exons 27 – 28 deletion
27
28
CMA Comprehensive
CMA +SNP
400 K CMA comprehensive showing normal CMA with a paternal UPD15
(Isodisomy)
Chromosome 15
Whole genome
Copy number plot
Agilent-SNP
In house array
Agilent-SNP
Genome Workbench
Illumina
CMA Comprehensive (180K + SNP Screen)
Normal CMA profile
AOH observed in close
relative mating
(example : father and
daughter mating)
Resolving Variants of
Uncertain Significance
Examples of Clone Plots
Current case
Nonpolymorphic
clone plot
Highly unusual
Current case
Polymorphic
clone plot
Common variant
Resolving Variants of Uncertain Significance
Mother
Father
Fetus
Chr 12 CNV
(paternal)
Trisomy 21
Incidental finding [from the father while absent from the fetus]
Proband
Mother
Father
Prenatal CMA
Considerations
Why Consider Prenatal CMA?
•
•
•
•
•
Combined incidence of known microdeletion/
duplication syndromes is at least ~1/1000
Many are not detected on standard chromosome
analysis, especially in prenatal samples with
lower resolution
Many have moderate to severe phenotypes after
birth, but no prenatal signs that would raise
suspicion and trigger specific FISH testing
Women of all ages are equally likely to have
affected pregnancies
No screening tools have been developed for
microdeletion / microduplication disorders
Baylor Prenatal CMA Clinical Protocol
• Parental samples required for testing
• Informed consent strongly recommended
• Entire sample can be sent to Baylor (30+cc amniotic
fluid {>16 wk gestation} or 30+ CVS) for routine
cytogenetics and CMA OR direct sample may be
split (15cc amniotic fluid or 15 mg dCVS) can be
sent and remainder sent to another lab for routine
cytogenetics.
• Direct CMA testing on direct CVS or direct amniotic
fluid with (collected >16 weeks gestation) with
culture in reserve.
• Maternal cell contamination studies
• Reporting in 7 - 10 days from direct sample
Pre-test discussion
A pre-test discussion should include:
– How the testing works
– What is being tested for
– Possible test results
– Benefits of testing
– Limitations/risks of testing
– Assessment of the individual’s understanding
of the testing
– Assessment of parental clinical and
developmental history
Possible Test Results
• No abnormality detected
– No gain or loss of chromosomal material was detected in
the regions tested
– A gain or loss was detected that is known / expected to be
benign (i.e. does not cause disease)
• Abnormality detected
– A gain or loss of chromosomal material known to result in
a defined genetic condition has been detected
• Results of uncertain significance
– A gain or loss of chromosomal material not known to
result in a defined genetic condition has been detected
– This means that a change was found, but there is little or
no medical knowledge about the particular change.
Whether the change may lead to medical problems and
what types of problems it may cause is uncertain. In this
case additional testing is performed, including analysis of
DNA from the parents.
Benefits of CMA testing
• CMA testing may discover an abnormality
that may not have been detected by
routine chromosome testing.
• The information gained from CMA testing
may be important for making decisions
about the pregnancy or for making medical
decisions about the baby’s care after
delivery.
Limitations of CMA testing
• Detection rates
– It is possible that the baby could have one of the medical conditions
included in the CMA test, but the CMA test was unable to detect the
condition.
– For some conditions included in the CMA test, 99% of cases can be
detected.
– For others, the detection rate may be lower because they can have
multiple underlying causes.
• Findings of uncertain significance
– It is possible that the test will detect an abnormality for which there
is very little medical information available to predict the type of
problems that may develop in the baby.
– Expectant parents may be left with ambiguity and this may increase
their anxiety about the pregnancy
• Need for further testing and impact on family members
– As with any genetic test, results may indicate a need for further
testing and may also impact other family members.
Assessment of Patient Understanding
Patients should understand that:
– CMA DOES NOT test for ALL genetic
conditions
– Detection rates are not 100%
– Even if the results are normal, the baby could
still have a birth defect(s) or mental
retardation from causes not detected by the
CMA testing
Prenatal CMA
Case Examples
Case 1 CVS (13 weeks)
Indication: AMA, Abnormal ultrasound (nuchal thickening)
and Normal chromosome analysis referred to us for CMA
8.7 Mb loss in 4q21.23q22.1
Copy number loss of this region is associated with CNS
overgrowth, facial anomalies, hypotonia and
developmental delay [PMID 9098490]
Case 1 CVS (13 weeks)
Confirmation of the deletion by FISH. In
retrospect, perhaps karyotype shows deletion.
Deleted
chromosome
Deleted
chromosome
Case 2
Indication: AMA, parental concern
•
Duplication detected at 17p11.2 involving
RAI1. Duplication of RAI1 has been
associated with Potocki-Lupski syndrome.
•
FISH using the probe FLI in green used in the
clinical laboratory confirmed an additional
copy of RAI1. The control probe PMP22 in red
shows the expected 2 signals.
Case 3: Origin of Marker Chromosome
Indication: Karyotype analysis performed at another
laboratory showed a supernumerary small marker
chromosome in 5 of 18 cells (28%). FISH studies with
probes specific for chromosomes 13, 14, 15, 18, 21, 22, X
and Y were unable to identify the origin of the marker.
• CMA detected a 10 Mb (maximum 19 Mb) gain in copy
number on the short arm of chromosome 20.
• Metaphase FISH analysis confirmed that the marker
chromosome was an isochromosome 20p resulting in
tetrasomy for 20p, and was observed in 20% (4/20) of
the cells examined.
Case 3: Origin of Marker Chromosome
Mar
20
20
Types of Cancer arrays
• HEME-ONC Array(44K)
• 180K CGH/SNP Cancer Array (CCMC
Design)
• BCM 400K CGH/SNP Cancer Array
Utility of Heme-Onc CMA
• First cancer gene targeted oligonucleotide
microarray for genome profiling of hematological
malignancies at a high resolution.
• Heme-Onc CMA is for:
–
–
–
–
–
Acute leukemias
CLL
Multiple myeloma
Lymphomas
Myelodysplastic syndrome (MDS)
• Higher sensitivity for detection of abnormalities
in CLL than the current FISH panel
Heme-Onc Array Design Guideline
Even Distribution
Target Genes Implicated
in Cancer
V.1.0
10X
Targeted Regions:
• Selected Genes (494)
• Regions Implicated in
Leukemia
X
Excluding Regions:
• Repetitive Elements
• Low Copy Repeats
• Assembly Gaps
• Copy Number Polymorphism
(TCAG V1+UCSC)
Heme-Onc Array Design Example
Gene 10X
Backbone Regions X
Resolution of the Heme-Onc Array
494 genes
1 OLIGO per 7.5 kb
Backbone region
1 OLIGO per 78 kb
Case 1: Indication – CLL
1. The initial chromosome study showed an abnormal clone
with deleted chromosome 11q and deleted chromosome
13q in 10% of the cells examined.
•
46,XY,del(11)(q13q23),del(13)(q14q22)[3]/46,XY[27]
2. These findings were consistent with the CMA results (blue
circles)
3. FISH confirmation showed that the deletions are present in
>40% of cells.
4. CMA also detected additional findings as indicated in
the pink box.
Case 1: Indication – CLL
2p16 gain
42% deleted by FISH
45% deleted by FISH
Case 2: Indication – CLL
1. Initial FISH results using CLL FISH panel
nuc ish(p53x1)[27/500] 5%
nuc ish(D13S319x0)[113/500] 27%
nuc ish(CEP12x3)[119/500] 27%
2. These findings were consistent with the CMA results
(blue circles)
3. CMA also detected additional findings as indicated in
the pink boxes
Case 2: Indication – CLL
dup15q
dup 11q
del 9p
Case 3: Indication – MDS
1. Initial chromosome analysis detected two abnormal clones
•
47,XX,inv(3)(q21q26),+mar[11]/46,idem,-7[9]
2. CMA detected a loss of chromosome 7 (red circle) except
for the pericentromeric region (blue arrow).
3. Subsequent FISH analysis using a centromere probe for
chromosome 7 confirmed the marker chromosome is
derived from chromosome 7.
4. CMA is able to detect gain or loss of genomic material but
not balanced rearrangements such as the inverted
chromosome 3 present in this case.
Case 3: Origin of the marker chromosome
inv(3)
marker
298857
56131915
Chr 7
95893139
mar derived
from chr 7
158767840
Case 4: Indication – MDS
Right Panel: Initial chromosome
analysis detected an abnormal
clone:
marker
46,XY,-7,+mar[11]/46,XY[10]
Bottom Panel: CMA detected a
gain of chromosome 3q and a loss
and gain of chromosome 7q.
chr 3
nl 7
Case 4: CMA Results continued
Marker chromosome is
an isoderivative 7
Chr 3
Chr 3
ider(7)(q22)t(3;7)(q25.3;q22)
Chr 7
Case 5: Indication-CLL- Homozygous loss on 13q14
A. CMA detected a LOSS of copy number (deletion) on
chromosome 13.
B. The chromosome 13-specific plot shows the
coverage and the boundaries of the deleted
segment.
C. The size of the deleted segment and genes involved.
D. The size of the segment within the deletion showing
a homozygous LOSS and the genes involved.
Case 5: Indication-CLL- Homozygous loss on 13q14
A.
B.
C. Copy number LOSS on chromosome 13
D. Homozygous loss
Case 5: Indication-CLL- Homozygous loss on 13q14
Confirmation FISH analysis using the CLL FISH panel from Vysis
E.
E. 63% (313/500) cells had one
signal for D13S319 (red) probe.
F.
F. 27% (146/500) cells had no
signals (homozygous loss) for
D13S319 probe localized to
chromosome 13q14 consistent
with the results from array
CGH.
13q14 – Red Signal
13q34 – Aqua Signal
12cen – Green Signal
180K CGH/SNP Cancer Array
(CCMC Design)
180K CGH/SNP Cancer Array (CCMC Design)
MLL
44K
60K
105K
180K
Backbone
DDX6
CBL2
Backbone
• 512 cancer genes or cancer-related genes
• Average of 2 probes per exon.
• Average resolutions <10 Kb (large exons) to <10 Kb
(cancer genomic regions) in targeted regions.
• ~30 Kb in backbone regions.
Myelodysplastic Syndrome
(180K CGH/SNP Array)
4 copies
3 copies
4
3
2
1 copy
0 copy
BBBB
ABBB
AAAB
AAAA
• Myelodysplastic Syndrome
•Partial Chromosome 6p Amplification to 4 copies
• SNP data show 0, 1, 3, 4 copies
1
0
Unbalanced “Balance Translocation”
Acute Myeloid Leukemia (180K CGH Array, CCMC Design)
Red: D7S486
Green: D7Z1
57 Mb
3p21.3
1 2.7
Mb
12p13.3
1 2.7 Mb
3q21.3
1.6 Mb
3q26.2
87 Kb
36 Mb
Red arrow: deletions; green arrow: duplication
BCM 400K CGH/SNP Cancer Array
BCM 400K CGH/SNP Cancer Array
MLL
DDX6
400K
Backbone
Backbone
• 2,300 cancer genes or cancer-related genes
• 235 cancer associated-miRNAs
• Average of 6 probes per exon.
• Average resolutions <1 Kb (large exons) to <10 Kb (cancer
genomic regions) in targeted regions.
• ~12 Kb in backbone regions.
Myelodysplastic Syndrome
(BCM 400K CGH/SNP Array)
Copy Gain LOH
3 copies
Triplication
of one allele
1 copy
Homozygous
deletion
Copy Loss LOH
3 copies
Triplication
of one allele
Copy Gain LOH
SNP
CGH
Conclusion
• CMA is a powerful molecular cytogenetic tool for
detecting genomic imbalances both in constitutional
as well as in cancer diagnostics
• CMA is a high resolution technology capable of
detecting chromosomal DNA copy number changes
throughout the genome
• Baylor designed CMA detects chromosomal
abnormalities that would go undetected by older
techniques such as karyotype or non-exon focused
arrays