Shashikant Kulkarni, M.S (Medicine)., Ph.D., FACMG
Head of Clinical Genomics
Medical Director of Cytogenomics and Molecular Pathology
Associate Professor of Pediatrics, Genetics, Pathology and Immunology
http://clinicalgenomics.wustl.edu
Dislosures
(compensated and non-compensated)
• Scientific Advisory Board/Consultant
– National Institute of General Medical Sciences (NIGMS)
Coriell cell repositories
– Chromosome Disorder Outreach
– Genomequest
– Agilent technologies
• Speaker honorarium
– National Cancer Institute (NCI), American College of
Medical Genetics, Association of Molecular Pathology,
University of Minnesota, University of Florida, CDC,
Molecular Medicine- tricon, OMICS revolution, Illumina,
Novartis, Affymetrix, Agilent
Genomics and Pathology Services at Washington University
Research & Panel
Development
Genomic
Technologies
and Innovation
Clinical
Genomics
Biomedical
Informatics
Pathology
Consulting
Services
Clinical
Genomics
Biomedical
Informatics
Training and
Education
 Over 150 faculty and staff support GPS function
 Computational biologists, bench scientists
 Software engineers, informaticians
 Biostatisticians, IT administrators
 Board-certified clinical genomocists and pathologists (ABMG,
ABP)
 ~15,000 sq ft dedicated labs; majority CAP/CLIA
 Chromosomes to base pairs
 Next generation sequencing
11/11/11: Clinical Launch – Cancer Panel
Clinical exome sequencing
Targeted assays for inherited disorders
Pharmacogenetic assays
Clinical Genomics
• Current state of diagnostic testing
– Constitutional
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Washington University School of Medicine, St Louis
Chromosomal microarrays
Karyotyping (?)/phenotype directed FISH tests (?)
Single gene molecular testing
Next Generation sequencing (NGS) disease panels
NGS- exome and whole genome sequencing
– Cancer
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FISH (rapid)
Karyotyping (whole genome view)
Chromosomal microarrays (?)
Single gene molecular testing
Next Generation sequencing (NGS) cancer specific panels
NGS- exome and whole genome sequencing
Clinical Genomics
• Chromosomal Microarrays (CMA)-aCGH and
SNP arrays
– Increasing our understanding of genomic
aberrations at very high resolution
• Next Generation sequencing (NGS)
– Revolutionizing, paradigm shifting look at the
genome
• Fluorescence in situ hybridization (FISH) is key
in integrated clinical genomic analyses
FISH
• Most common method for verifying CMA findings
• Visualization in intact cellular context
– Positional and orientational information of
chromosome structures
• Rapid turn around time
• Better detection of low-level mosaicism
• Best method for rapid detection of translocations,
inversions, amplifications, deletions and
duplications
• CMA and Next generation sequencing still lacks
most of the above
FISH
• Clone based methods
• FISH probes typically generated from genomic
DNA, bacterial artificial chromosomes (BAC),
fosmids, PAC (P1-derived artificial chromosome),
YAC (yeast artificial chromosome), PCR templates
• Most FISH probes are between 150-300 Kb
• Not useful for visualization/verification of smaller
abnormalities
• PCR template generated probes
– Not ideal, time consuming, multiple steps
Cases to demonstrate limitations
of clone based FISH methods
Case-1
• Three year old boy with global developmental
delay, hypotonia, speech problems
• Chromosomal Microarray (CMA) reveals ~70Kb
deletion on 6q22.33 disrupting LAMININ, ALPHA2; (LAMA2)
– Laminin is a heterotrimeric extracellular matrix protein
consisting of 3 chains: alpha-1, beta-1, and gamma-1.
– Several isoforms of each chain have been identified.
Laminin-2 (merosin) is a heterotrimer composed of
laminin subunits alpha-2, beta-1, and gamma-1.
– It is the main laminin found in muscle fibers
Case-1
• Clone based FISH methods failed to help
verify/visualize LAMA2 deletion
– BAC, fosmids
• Parental studies performed by CMA
• Deletion maternal in origin
• Mutation of the other allele
Case-2
A 39 year-old woman with acute myeloid leukemia (AML)
referred for an allogeneic stem cell transplant
Atypical promyelocytes with invaginated nuclei
(dense primary granules)
Does the patient have APL, or does she have AML with
unfavorable-risk cytogenetics?
RARA-PML fusion
Impact of NGS on Cancer Genomics
Schematic representation of ins(15;17)
identified by WGS and resulting in
PML-RARA fusion
FISH identifies a fusion event on
der(17), consistent with ins(15;17)
et al
Oligonucleotide FISH
• Uses high complexity oligonucleotide libraries
as starting point for probe generation
• Bioinformatic approaches and various
algorithms are used for probe selection based
on in silico predictions
• Repetitive elements can be avoided
• Precise genomic coordinates from reference
genomes are used to generate probes
Oligonucleotide FISH
• Involved in early strategic design and selection
of probes utilizing knowledge generated by
genome sequencing
• Understanding of detailed genomic structure
very useful in carefully excluding noise
generating repetitive elements
• Preliminary experimental data presented here
on results from pilot research studies
Oligos specifically selected to unique
sequences
Repetitive elements
Step one: tile region with long oligonucelotides:
Segmental duplications
Step two: Remove any non-unique oligos:
Step Three: Manufacture labeled probes using specifically designed long oligonucleotides
Advantages of Oligo FISH Over BACFISH
Oligo FISH
BAC-FISH
Minimum region targeted
<50kb
~100kb
Requires available clone?
No
Yes
Need Cot-1 DNA ?
No
Yes
Can specifically target regions
that have a high degree of
homology/repetitive
elements?
Yes
No
Probe Signal to noise
+++
++
Detect chromosome
rearrangements?
Yes
Yes
4-14 hours
~14 hours
Hybridization time
Detection of Smaller Regions
Repeat Gaps
c-met locus divided into 6 regions
Region 1
Sequence
Tiled (kb)
1
23.3
14.1
2
20.0
14.1
3
27.9
14.1
4
27.6
14.1
5
31.4
14.1
6
23.6
13.6
Region 2
Red : SureFISH probe
Green: BAC CEP
Regions 1-6
20/20 metaphase and 20/20 interphase
cells showed this staining
Region
Size (kb)
Region 3
Region 5
Region 4
Region 6
Detection of Difficult Regions
Region
Size
% Repeat
Num Gaps
Median Gap Size
Max Gap
Size
Tiled Region
% GC
23 kb
61%
5
660bp
1.2 kb
8.6kb
62%
Green: BAC CEP
Red: SureFISH Probe
A 6.7-kb region at 6p22.2 (110,219,652–110,316,643)
is detected using oligonucleotide-based FISH, shown
by the red signals.
The same FISH image is shown with DAPI
counterstain (left), and inverted DAPI stain or ‘pseudo G-banding’
confirming the chromosomal location (right). Arrows indicate
chromosome 6
Probe region selection – 1q21 region
• 3 different probes designed within region
between segmental duplications
• designed to cover genes in the regions
SureFISH probes
GeneTracks
Segmental
Duplications
Probe design – 1q21region
• Focus on 224kb region of interest
• Oligos in region target specific sequences
SureFISH probe location
Oligo coverage
within probe
GeneTracks
Segmental
Duplications
Repeat Masked Region
Research Pilot study
• OFISH (4 hour) compared to overnight BAC
based FISH
• OFISH performed on cases with smaller
genomic aberrations not easily detected by
chromosomal microarray (CMA)
• Preliminary data
BCR ABL POSITIVE
O-FISH
BCR=Red; ABL=Green
BAC probe
BCR=Green; ABL=Red
BCR ABL NEGATIVE
O-FISH
BAC probe
BCR=Red; ABL=Green
BCR=Green; ABL=Red
PML RARA POSITIVE
O-FISH
PML=Green; RARA=Red
BAC probe
PML=Red; RARA=Green
PML RARA NEGATIVE
O-FISH
PML=Green; RARA=Red
BAC probe
PML=Red; RARA=Green
CEP 8 POSITIVE
Trisomy
O-FISH
BAC probe
CEP 8 NEGATIVE
O-FISH
BAC probe
EGR1 POSITIVE
EGR1=Red; CEP5=Green
O-FISH
BAC probe
EGR1 NEGATIVE
EGR1=Red; CEP5=Green
O-FISH
BAC probe
D7S486 POSITIVE
D7S486 NEGATIVE
D7S486=Red; CEP7=Green
D7S486=Red; CEP7=Green
O-FISH
BAC probe
O-FISH
BAC probe
MLL POSITIVE
O-FISH
3’ Green; 5’ Red
BAC probe
3’ Red; 5’ Green
MLL NEGATIVE
O-FISH
3’ Green; 5’ Red
BAC probe
3’ Red; 5’ Green
O-FISH for RP11-414N15 Region
Chr15:31775207-31899230
Deletion
No Cross-hybridization
Control
O-FISH for RP11-433J22 Region
Chr1:147162143-147386375
Duplication
duplicated signal
Control
Preliminary results
• Signal intensity, sensitivity, specificity,
reproducibility of OFISH probes determined
• High intensity, robust signals could be
generated from regions that are smaller to
detect by clone based methods
• 100% concordance between clone based FISH
methods and OFISH
• 100% concordance between CMA findings and
OFISH
Summary
• OFISH is a powerful alternative to clone based FISH
methods
• Use of genomic information to design probes helps in
generation of highly reproducible robust FISH probes
• Additional studies are underway
• Ability to detect smaller aberrations not previously
visualized by traditional FISH probes is very valuable as
we enter the high resolution, fine scale clinical
genomics era
• Availability of OFISH probes with sequence level
information and confirmation of chromosomal
localization and performance quality metrics will
markedly improve study of genome complexities
Karen Seibert, John Pfiefer, Skip Virgin,
Jeffrey Millbrandt, Rob Mitra, Rich Head
Rakesh Nagarajan and his Bioinf. team
David Spencer, Eric Duncavage, Andy Bredm.
Hussam Al-Kateb, Cathy Cottrell
Dorie Sher, Jennifer Stratman
Tina Lockwood, Jackie Payton
Mark Watson, Seth Crosby, Don Conrad
Andy Drury, Kris Rickoff, Karen Novak
Mike Isaacs and his IT Team
Norma Brown, Cherie Moore, Bob Feltmann
Heather Day, Chad Storer, George Bijoy
Dayna Oschwald, Magie O Guin, GTAC team
Jane Bauer and Cytogenomics &Mol path team
MANY MORE!