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SNP OligonucleotideMicoroarray Analysis
(SOMA)in Clinical Cytogenetics
Brynn Levy, M.Sc.(Med)., Ph.D.
• The best whole genome analysis technique currently available is …………………..
GTG-banding of Metaphase chromosomes
Chromosome Analysis
Chromosome Analysis
• G-banding provides a visual examination of the entire genome
• It therefore provides the best coverage but not the best resolution
• Banding resolution differs from preparation to preparation
Diagnostic Limits of Conventional Cytogenetic Analysis
• “Obvious” Aneuploidies & Rearrangements should be easily diagnosed
• The smaller the region of gain/loss, the harder it is to detect
• At Absolute best, imbalances in the realm of 2-5Mb may be detected� Most banding resolutions will allow detection of
gains/deletions of >5Mb
• Absolute best is dependent on banding resolution
How Does Banding Resolution Impact Diagnostic Ability ?
• Small & Subtle aberrations may be missed� Depends on Banding Resolution & Specimen Preparation
• Most clinical labs strive for 550 band level resolution in postnatal studies and greater than 400 bands in prenatal studies. The resolution in bone marrows and products of conception are often at or below the 400 band level
• Make do with what you have and accept the limitations
How Does Banding Resolution Impact Diagnostic Ability ?
Molecular Cytogenetics
• Application of the techniques of Molecular Biology to cytogenetic preparations
• Molecular cytogenetic techniques provide a way to detect complicated, cryptic and submicroscopic rearrangements that remain undetected or undecipherable by conventional cytogenetic analysis
15.3
15.1
14
13.3
13.1
12
11.1
11.2
12
13.1
13.3
14
15
21
22
23.1
23.3
31.1
31.2
31.3
32
33.1
33.3
34
35.1
35.3
15.3
15.1
14
13.3
13.1
12
11.1
11.2
12
13.1
13.3
14
15
21
22
23.1
23.3
31.1
31.2
31.3
32
33.1
33.3
34
35.1
5 add(5)(q35)
Partial Karyotype of Patient
5 add (5)(q35)
wcp 11wcp 5
Chromosome 11
Chromosome 5
Microdeletion Studies Using FISH
Syndrome Chromosome Location Probe/Gene Locus
DiGeorge 22q11.2 D22S75
Velocardiofacial 22q11.2 D22S76
Miller-Dieker 17p13.3 D17S379
Smith-Magenis 17p11.2 D17S29
Prader-Willi 15q11.2 SNRPN
Angelman 15q11.12 D15S10
Williams 7q11.23 Elastin
Cri du chat 5p15.2 D5S23
Wolf-Hirschhorn 4p16.3 D4S96
Prader-WilliSyndrome ??
The Clinical Phenotype Guides the Choice of FISH Test
Williams Syndrome ??
The Clinical Phenotype Guides the Choice of FISH Test
What FISH test do we do in this case ???
Detection of Partial Aneuploidies –An expensive FISHing Expedition
• Unbalanced rearrangements
• Marker chromosomes
• Cryptic translocations
• Cryptic deletions
• Suspected Microdeletions with non-
specific clinical abnormalities
The Need for New TechnologiesThe Need for New TechnologiesThe Need for New TechnologiesThe Need for New Technologies
COMPARATIVE GENOMIC HYBRIDIZATION
C G H
• Identifies chromosomal gains and losses in a
single hybridization procedure
• Effectively reveals any DNA sequence copy number changes (i.e., gains, amplifications, lossesand deletions) in a particular specimen and maps
these changes on normal chromosomes
q21q22
Initial Karyotypic Designation46,XY,t(1;2)(p22;q14.1)
CGH in a patient with an “Apparently Balanced Translocation” and Clinical Abnormalities
2P Telomere
2q Telomere
BAC RPCI-11 91A11
2P Telomere
2q Telomere
BAC RPCI-11 91A11
Interpreting a Normal CGH Result in a Karyotypically Normal Individual with Clinical
Abnormalities
• A normal CGH result has to be interpreted within
the boundaries of the test’s limitations
• A normal CGH result does NOT rule out balanced
cryptic rearrangements
• A normal CGH result does NOT rule out sub-
microscopic imbalances such as microdeletions
Case #: D8 Del 2 M4 M6 D6 D9
Approx. 6-7Mb 14-15Mb 14-16Mb 18-20Mb 9-11Mb 3-4Mb
size of
imbalance
2q+ 2 4 4q- 6 mar 15 mar 22q+ 22 19 mar
CGH IN CLINICAL CYTOGENETICS
• Precise identification of extra or missing material– Important for diagnostic and prognostic value– Important for identifying those genes causative of the
clinical phenotype
• Single step global genome scan preventsFISHing expedition
• DNA based analysis– Quality of patient metaphase spreads is not a consideration
– Non-viable tissues are amenable to analysis
Arrayed clonesArrayed clones
Cloned humanCloned humanDNA (BAC/PAC) DNA (BAC/PAC)
CGH Microarray MethodologyCGH Microarray Methodology
Control genomic Control genomic DNADNA
Test genomic Test genomic DNADNA
Automated Automated analysisanalysis
CGH Microarray MethodologyTrisomy 21 (47,XY,+21)
CGH Microarray Methodology
SNP Oligonucleotide Microarray Analysis (SOMA) in Clinical Cytogenetics – Preliminary Results
• SNP Oligonucleotide microarray for clinical cytogenetic analysis (SOMA)
• With over half a million SNPs and a mean spacing of 5.8 kb, the 500K SNP array offers a comprehensive whole genome scan and has the potential to provide the highest resolution of copy number detection currently available.
• Since deletions and duplications can be precisely defined by using the SNP positions on the genome browser, SOMA will useful for the clinical interpretation of both visible and submicroscopic cytogenetic imbalances.
• Another advantage of using a SNP-based array lies in the concurrent availability of genotype information that would allow simultaneous DNA-based studies such as uniparental disomy (UPD), zygosity and maternal cell contamination.
• Cases were chosen to contain a variety of known cytogenetic aberrations, including whole chromosome aneuploidy, unbalanced rearrangements, marker chromosomes and microdeletions.
• Copy number gains & losses were determined by means of the Circular Binary Segmentation method.
The Use of High Resolution OligonucleotideMicroarray Methodology in Clinical Cytogenetics
Four Important Diagnostic Categories
1. Aneuploidy� Trisomies & Monosomies
2. Partial Aneuploidy at or just below the threshold of the maximum resolution of G-banding (>1-5Mb)
� Unbalanced translocations (of unknown origin)
� Intrachromosomal deletions/duplications
� Marker chromosomes
� Unbalanced “balanced rearrangements”
� Chromosomal rearrangements of indiscernible nature
3. Known Microdeletions/Microduplications (Dynamic list) and Subtelomericimbalances
� Prader-Willi
� DiGeorge
� Cryptic unbalanced translocations
� Terminal Deletions/Duplications
4. Partial Aneuploidies below the resolution of G-banding (<1Mb)� New clinically significant microdeletions/microduplications
Category 1 Cases- Aneuploidy
NormalNoneNone46,XXNormal Reference
Trisomy 21NoneNone47,XX,+21r/o Down Syndrome
NormalNoneNone46,XXNormal Reference
NormalNoneNone46,XYNormal Reference
SOMAAdditional
Study
Results
Workup Additional
KaryotypeIndication
Category 2 Cases - Partial Aneuploidies (>1-5Mb)
47,XX,+r
47,XX,+r
47,XX,+r
Category 2 Cases
8.5
1.8
dim(13)(q14.3q21.2)
dim(13)(q22.1)
dim(13)(q14.3q21.2)
dim(13)(q22.1)ROMA & FISH
46,XY,t(3;10)(q23;q11.2),del(13)
(q14.3q21.2)
Mild Phen
9.4
6.8
2
6.8
11.3
17.3
17.1
15.2
SOMA
Imbalance
(Mb)
enh(15)(q26.1q26.3)enh(15)(q24q26.2)CGH & FISH47,XY,+marSGS
dim(8)(p23.1p23.3)
dim(13)(q11.1q12.11)
enh(8)(p21.2p23.1)
dim(8)(p23.3pter)
dim(13)(q12)
enh(8)(p21p23)
CGH & FISH46,XX,t(8;13)MCA/MR
dim(18)(q12.1q12.3)dim(18)(q12q21)der(18)del(18)(q12q21)t(3;18)(p26;q21.3)
CGH46,XX,t(3;18)MCA/MR
enh(15)(q25.2q26.3)
enh(15)(q25.3q26.3)enh(15)(q24qter)CGH & FISH47,XX,+marSGS (x2)
enh(4)(p12q13.1)enh(4)(p11q13.1)CGH & FISH47,XX,+rMCA/MR
SOMAAdditional
Study Results
Additional
WorkupKaryotypeIndication
Shprintzen-Goldberg Like Phenotype
Patient at age 4½
Cytogenetic Findings
Amniocentesis:
• Revealed mosaicsm for a small marker
chromosome
47,XX,+mar
Molecular Cytogenetic Findings
Postnatal:
• Painting with multiple whole chromosome paints (wcp) revealed the marker to be derived from chr. 15
• PW/AS probe negative� Marker lacks 15 centromeric alpha satellite� Marker does not contain 15q11-q13 region (PW/AS)� Marker does not contain 15q22 region (PML)
• Molecular cytogenetic analysis using Comparative Genomic Hybridization (CGH) showed an overrepresentation of 15q24-qter, consistent with tetrasomy of this region
• Tetrasomy confirmed using a subtelomeric probe for 15q
• The marker was positive for CENP-C indicating the presence of a functional centromere and thus confirming the presence of a neocentromere
wcp 3
wcp 15
Neo 15q – CENP C & Alpha Satellite
15 qtelLSI PML
Category 2 Cases
8.5
1.8
dim(13)(q14.3q21.2)
dim(13)(q22.1)
dim(13)(q14.3q21.2)
dim(13)(q22.1)ROMA & FISH
46,XY,t(3;10)(q23;q11.2),del(13)
(q14.3q21.2)
Mild Phen
9.4
6.8
2
6.8
11.3
17.3
17.1
15.2
SOMA
Imbalance
(Mb)
enh(15)(q26.1q26.3)enh(15)(q24q26.2)CGH & FISH47,XY,+marSGS
dim(8)(p23.1p23.3)
dim(13)(q11.1q12.11)
enh(8)(p21.2p23.1)
dim(8)(p23.3pter)
dim(13)(q12)
enh(8)(p21p23)
CGH & FISH46,XX,t(8;13)MCA/MR
dim(18)(q12.1q12.3)dim(18)(q12q21)der(18)del(18)(q12q21)t(3;18)(p26;q21.3)
CGH46,XX,t(3;18)MCA/MR
enh(15)(q25.2q26.3)
enh(15)(q25.3q26.3)enh(15)(q24qter)CGH & FISH47,XX,+marSGS (x2)
enh(4)(p12q13.1)enh(4)(p11q13.1)CGH & FISH47,XX,+rMCA/MR
SOMAAdditional
Study Results
Additional
WorkupKaryotypeIndication
Neocentric Chr 15FISH the Breakpoints
Balanced t(8;13) ???
8p
8q
CEP 13/21
13 8derivative 8
Proposed Sequence of Events
Category 2 Cases
8.5
1.8
dim(13)(q14.3q21.2)
dim(13)(q22.1)
dim(13)(q14.3q21.2)
dim(13)(q22.1)ROMA & FISH
46,XY,t(3;10)(q23;q11.2),del(13)
(q14.3q21.2)
Mild Phen
9.4
6.8
2
6.8
11.3
17.3
17.1
15.2
SOMA
Imbalance
(Mb)
enh(15)(q26.1q26.3)enh(15)(q24q26.2)CGH & FISH47,XY,+marSGS
dim(8)(p23.1p23.3)
dim(13)(q11.1q12.11)
enh(8)(p21.2p23.1)
dim(8)(p23.3pter)
dim(13)(q12)
enh(8)(p21p23)
CGH & FISH46,XX,t(8;13)MCA/MR
dim(18)(q12.1q12.3)dim(18)(q12q21)der(18)del(18)(q12q21)t(3;18)(p26;q21.3)
CGH46,XX,t(3;18)MCA/MR
enh(15)(q25.2q26.3)
enh(15)(q25.3q26.3)enh(15)(q24qter)CGH & FISH47,XX,+marSGS (x2)
enh(4)(p12q13.1)enh(4)(p11q13.1)CGH & FISH47,XX,+rMCA/MR
SOMAAdditional
Study Results
Additional
WorkupKaryotypeIndication
Category 2 Cases
8.5
1.8
dim(13)(q14.3q21.2)
dim(13)(q22.1)
dim(13)(q14.3q21.2)
dim(13)(q22.1)ROMA & FISH
46,XY,t(3;10)(q23;q11.2),del(13)
(q14.3q21.2)
Mild Phen
9.4
6.8
2
6.8
11.3
17.3
17.1
15.2
SOMA
Imbalance
(Mb)
enh(15)(q26.1q26.3)enh(15)(q24q26.2)CGH & FISH47,XY,+marSGS
dim(8)(p23.1p23.3)
dim(13)(q11.1q12.11)
enh(8)(p21.2p23.1)
dim(8)(p23.3pter)
dim(13)(q12)
enh(8)(p21p23)
CGH & FISH46,XX,t(8;13)MCA/MR
dim(18)(q12.1q12.3)
dim(18)(q12q21)
der(18)del(18)(q12q21)t(3;18)(p26;q21.3)
CGH46,XX,t(3;18)MCA/MR
enh(15)(q25.2q26.3)
enh(15)(q25.3q26.3)enh(15)(q24qter)CGH & FISH47,XX,+marSGS (x2)
enh(4)(p12q13.1)enh(4)(p11q13.1)CGH & FISH47,XX,+rMCA/MR
SOMAAdditional
Study Results
Additional
WorkupKaryotypeIndication
Patient Clinical Info
Delayed major motor milestones, including rolling over at 4 months, sitting without support at 8 months, and walking at 22 months. He had been receiving regular physical therapy for delay in gross motor skills. He spoke in short sentences and understood complex commands. There were no facial dysmorphisms besides slightly cupped ears and no medical problems except for severe eczema.
Normal development status at 3 yrs, except for mild delay in gross motor skills and coordination.
46,XY,t(3;10)(q23;q11.2),del(13)(q14.3q21.2)
N_AB
0
1
2
3
4
5
6
7
0 20000000 40000000 60000000 80000000 100000000 120000000
CNAG 2.0
MSSM
Data AnalysisROMA
Category 2 Cases
8.5
1.8
dim(13)(q14.3q21.2)
dim(13)(q22.1)
dim(13)(q14.3q21.2)
dim(13)(q22.1)ROMA & FISH
46,XY,t(3;10)(q23;q11.2),del(13)
(q14.3q21.2)
Mild Phen
9.4
6.8
2
6.8
11.3
17.3
17.1
15.2
SOMA
Imbalance
(Mb)
enh(15)(q26.1q26.3)enh(15)(q24q26.2)CGH & FISH47,XY,+marSGS
dim(8)(p23.1p23.3)
dim(13)(q11.1q12.11)
enh(8)(p21.2p23.1)
dim(8)(p23.3pter)
dim(13)(q12)
enh(8)(p21p23)
CGH & FISH46,XX,t(8;13)MCA/MR
dim(18)(q12.1q12.3)
dim(18)(q12q21)
der(18)del(18)(q12q21)t(3;18)(
p26;q21.3)CGH46,XX,t(3;18)MCA/MR
enh(15)(q25.2q26.3)
enh(15)(q25.3q26.3)enh(15)(q24qter)CGH & FISH47,XX,+marSGS (x2)
enh(4)(p12q13.1)enh(4)(p11q13.1)CGH & FISH47,XX,+rMCA/MR
SOMAAdditional
Study Results
Additiona
l WorkupKaryotypeIndication
Detailed Examination of Deleted Regions
Category 2 Cases - Cont
0.7dim(15)(q11.2)SNRPN-FISH47,XY,+marSpeech delay
Mild Developmental
delay
7.5
8.4
7.4
19.1
SOMA
Imbalance
(Mb)
dim(2)(q23.1q24.1)dim(2)(q23?q24)CGH & FISHNormalMCA/MR
dim(2)(q23.1q23.3)--46,XX,del(2q37)MCA/MR
dim(11)(p12p14.1)BAC deletionFISH46,XX,del(11p13)Aniridia, MR & ambiguous genetalia
dim(18)(q21.32-18q23)
--del(18q21.32)MCA/MR
SOMA
Additional
Study
Results
Additional
WorkupKaryotypeIndication
Category 2 Cases - Cont
0.7dim(15)(q11.2)SNRPN-FISH47,XY,+marSpeech delay
Mild Developmental
delay
7.5
8.4
7.4
19.1
SOMA
Imbalance
(Mb)
dim(2)(q23.1q24.1)dim(2)(q23?q24)CGH & FISHNormalMCA/MR
dim(2)(q23.1q23.3)--46,XX,del(2q37)MCA/MR
dim(11)(p12p14.1)BAC deletionFISH46,XX,del(11p13)Aniridia, MR & ambiguous genetalia
dim(18)(q21.32-18q23)
--del(18q21.32)MCA/MR
SOMA
Additional
Study
Results
Additional
WorkupKaryotypeIndication
Category 3 Cases
46,XX – Developmental Delay/CHD
46,XX – Developmental Delay/CHD
FISH Using DG Probe
Category 3 Cases
1.0
4.6
2.5
2.5
SOMA
Imbalance
(Mb)
dim(22)(q11.21)dim(22)(q11.2)FISHNormalDiGeorge
dim(4)(p16.2p16.3)WHS x1FISHNormalWHS
dim(9)(q34.3)9qter x 1FISHNormalMCA/MR
dim(22)(q11.21)dim(22)(q11.2)CGH & FISHNormalDiGeorge
SOMAAdditional
Study Results
Additional
WorkupKaryotypeIndication
Category 4 Cases
46,XX,t(11;22)(q23.3;p12)
Molecular Cytogenetic Findings
• CGH analysis normal
• FISH with putative breakpoint gene – normal
• SOMA - dim(20)(q11.22q11.23) – 3.7MB
Category 4 Cases
0.075-0.23
3.7
SOMA
Imbalance
(Mb)
Normal/CNVs/Gene dessertsTUPLE x2
10p probe x2FISHNormalDiGeorge
dim(20)(q11.22q11.23)NormalFISH46,XX,t(11;22)(q23.3;p12)
IUGR, dev delay,
and infantile
spasms
SOMAAdditional
Study
Results
Additional
WorkupKaryotypeIndication
Category 4 Cases
0.075-0.23
3.7
SOMA
Imbalance
(Mb)
Normal/CNVs/Gene desserts
TUPLE x2
10p probe x2FISHNormalDiGeorge
dim(20)(q11.22q11.23)NormalFISH46,XX,t(11;22)(q23.3;p12)
IUGR, dev delay,
and infantile
spasms
SOMAAdditional
Study Results
Additional
WorkupKaryotypeIndication
Oligonucleotide Microarrays
• Oligo Microarrays offer the greatest resolution� Raises the issue of copy number polymorphisms
− Their incidence– De novo versus familial
− Their clinical significance– Pathogenic versus benign variant– Association with disease
• Do they have any influence on the severity of disease
� Future population studies are needed− Ethnic / Race specific
� Improved software for determination of Copy Number− Integration of ongoing data from CNV studies to improve coverage area
Conclusion
• All previously characterized cytogenetic aberrations
could be identified using SOMA
• SOMA fine tunes the breakpoints
• SOMA gives a better assessment of the size of the
imbalance
• The genes involved in the imbalance can be determined
using the SNP positions on the genome browser thus
laying the foundation for more accurate genotype-
phenotype correlations
AcknowledgementsAcknowledgements
Columbia University Medical Center
• Vaidehi Jobanputra
• Odelia Nahum
• Wendy Chung
• Kwame Anyane-Yeboa
• Dorothy Warburton
Mount Sinai School of Medicine
• Y. Sun
• E. Bottinger
• W. Zhang Columbia UniversityMedical Center