snp oligonucleotide microarray analysis (soma) in clinical...

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

• 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

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

• 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

Results

Workup Additional

KaryotypeIndication

Category 2 Cases - Partial Aneuploidies (>1-5Mb)

47,XX,+r

Category 2 Cases

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

Imbalance

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 15

Neo 15q – CENP C & Alpha Satellite

15 qtelLSI PML

Category 2 Cases

dim(13)(q14.3q21.2)

dim(13)(q22.1)

dim(13)(q14.3q21.2)

46,XY,t(3;10)(q23;q11.2),del(13)

(q14.3q21.2)

Mild Phen

Imbalance

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)

enh(15)(q25.2q26.3)

SOMAAdditional

Study Results

Additional

Neocentric Chr 15FISH the Breakpoints

Balanced t(8;13) ???

CEP 13/21

13 8derivative 8

Proposed Sequence of Events

Category 2 Cases

dim(13)(q14.3q21.2)

dim(13)(q22.1)

dim(13)(q14.3q21.2)

46,XY,t(3;10)(q23;q11.2),del(13)

(q14.3q21.2)

Mild Phen

Imbalance

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)

enh(15)(q25.2q26.3)

SOMAAdditional

Study Results

Additional

Category 2 Cases

dim(13)(q14.3q21.2)

dim(13)(q22.1)

dim(13)(q14.3q21.2)

46,XY,t(3;10)(q23;q11.2),del(13)

(q14.3q21.2)

Mild Phen

Imbalance

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)

dim(18)(q12.1q12.3)

dim(18)(q12q21)

der(18)del(18)(q12q21)t(3;18)(p26;q21.3)

enh(15)(q25.2q26.3)

SOMAAdditional

Study Results

Additional

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)

0 20000000 40000000 60000000 80000000 100000000 120000000

CNAG 2.0

Data AnalysisROMA

Category 2 Cases

dim(13)(q14.3q21.2)

dim(13)(q22.1)

dim(13)(q14.3q21.2)

46,XY,t(3;10)(q23;q11.2),del(13)

(q14.3q21.2)

Mild Phen

Imbalance

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)

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)

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

Imbalance

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

Additional

Results

Additional

Category 2 Cases - Cont

0.7dim(15)(q11.2)SNRPN-FISH47,XY,+marSpeech delay

Mild Developmental

Imbalance

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

Additional

Results

Additional

Category 3 Cases

46,XX – Developmental Delay/CHD

FISH Using DG Probe

Category 3 Cases

Imbalance

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

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

Imbalance

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

Results

Additional

Category 4 Cases

0.075-0.23

Imbalance

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

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

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