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

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

Pathology Consulting Services

Biomedical

Informatics

Clinical Genomics

• Current state of diagnostic testing

– Constitutional

• Chromosomal microarrays

• Karyotyping (?)/phenotype directed FISH tests (?)

• Single gene molecular testing

• Next Generation sequencing (NGS) disease panels

• NGS- exome and whole genome sequencing

– Cancer

• 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

Washington University School of Medicine, St Louis

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, ALPHA-2; (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

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)

Impact of NGS on Cancer Genomics

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

Step one: tile region with long oligonucelotides:

Step two: Remove any non-unique oligos:

Repetitive elements

Segmental duplications

Step Three: Manufacture labeled probes using specifically designed long oligonucleotides

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

Hybridization time 4-14 hours ~14 hours

Advantages of Oligo FISH Over BAC-

FISH

Detection of Smaller Regions Region

Size (kb)

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 c-met locus divided into 6 regions

Repeat Gaps

Region 6

Region 2 Region 1

Red : SureFISH probe

Green: BAC CEP

Region 3

Region 5

Region 4

Regions 1-6 20/20 metaphase and 20/20 interphase cells showed this staining

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

GeneTracks

Segmental Duplications

Oligo coverage within probe

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 NEGATIVE

O-FISH BAC probe BCR=Red; ABL=Green BCR=Green; ABL=Red

BCR ABL POSITIVE

O-FISH BAC probe BCR=Red; ABL=Green BCR=Green; ABL=Red

PML RARA POSITIVE

O-FISH BAC probe PML=Green; RARA=Red PML=Red; RARA=Green

PML RARA NEGATIVE

O-FISH BAC probe PML=Green; RARA=Red PML=Red; RARA=Green

CEP 8 POSITIVE Trisomy

O-FISH BAC probe

CEP 8 NEGATIVE

O-FISH BAC probe

EGR1 POSITIVE

O-FISH BAC probe

EGR1=Red; CEP5=Green EGR1 NEGATIVE

O-FISH BAC probe

EGR1=Red; CEP5=Green

D7S486 POSITIVE

O-FISH BAC probe

D7S486=Red; CEP7=Green

D7S486 NEGATIVE

O-FISH BAC probe

D7S486=Red; CEP7=Green

MLL NEGATIVE

O-FISH BAC probe 3’ Green; 5’ Red 3’ Red; 5’ Green

MLL POSITIVE

O-FISH BAC probe 3’ Green; 5’ Red 3’ Red; 5’ Green

O-FISH for RP11-414N15 Region

Deletion Control No Cross-hybridization

Chr15:31775207-31899230

O-FISH for RP11-433J22 Region

Duplication Control

Chr1:147162143-147386375

duplicated signal

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!