4/19/2012
Genetics and Genomics in Clinical Research Bruce R. Korf, M.D., Ph.D. Department of Genetics Heflin Center for Genomic Sciences
Disclosures • Medical Director, UAB Medical Genomics Laboratory • Chair, Medical Affairs Committee, Children’s Tumor Foundation • Grant Funding: NIH, Department of Defense, Novartis • Advisory Boards: Novartis NF Advisory Board, March of Dimes
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Abby
Seth
Laura
Newborn Screening Diagnostic Prenatal Preconceptional Presymptomatic Predispositonal
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Newborn Screening Shortly after birth, blood is taken from Laura’s heel and sent to the State Newborn Screening Laboratory. Her parents are told that this is a routine test. No problems are found, and no follow‐up is needed.
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“We found an unexpectedly high proportion of literature‐annotated disease mutations that were incorrect, incomplete, or common polymorphisms.”
Immobilization/Amplification
Metzker, M. Nature Reviews Genetics 2010;11:3146.
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DNA Synthesis/Detection
Metzker, M. Nature Reviews Genetics 2010;11:3146.
Diagnostic Testing Laura is now 3 and her brother Seth is 5. Seth has been experiencing developmental problems, and is diagnosed as having autism
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Genetic Evaluation in Autism
Array CGH UAB Cytogenetics Laboratory Reference DNA
Test DNA
19.3 %
Mix Block repeated sequences
7.8 % 19.2 % 19.6 % 4.8 %
Hybridize Microarray with oligonucleotides
7.7 %
12.1 % 12.7 %
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The Diagnostic Odyssey Clinical problem
Try again
Differential diagnosis
Interpretation
Genetic testing
Genomic Diagnosis
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Genome Annotation
Secondary Findings
!
!
Green R, et al. Genetics in Medicine (2012) 14, 405–410
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Preconceptional Testing Laura is now married. She and her husband are considering starting a family and meet with her obstetrician‐gynecologist. They are both of Northern European ancestry and are offered carrier testing for cystic fibrosis.
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CF Carrier Screening
Ashkenzi Jewish
African/Asian/Mediterranean
Panethnic
Tay‐Sachs disease
Cystic fibrosis Cystic fibrosis
Canavan disease
Familial dysautonomia
Hemoglobinopathies
Spinal muscular atrophy
Inquire: •Mucolipidosis IV •Niemann‐Pick A •Fanconi anemia C •Bloom syndrome •Gaucher disease
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Genomic Carrier Testing
Prenatal Testing Laura and her Tom are indeed found to both be cystic fibrosis carriers. They elect to have prenatal diagnosis by amniocentesis at 16 weeks of pregnancy. The fetus is found to be a CF carrier.
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Prenatal Diagnosis
www.repromed.ca/pgd_toronto.html Dorland's Medical Dictionary for Health Consumers. © 2007 by Saunders, an imprint of Elsevier, Inc. All rights reserved.
amniocentesis
www.daviddarling.info/encyclopedia/C/chorionic_villus_sampling.html
chorionic villus biopsy
preimplantation diagnosis
Next Generation Prenatal Screening
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Genomic Prenatal Diagnosis
Presymptomatic Testing Laura is now 45. She has just learned that her older sister Abby, age 49, has been diagnosed as having breast cancer. She is concerned about her own risks, given that there is a family history of others with breast cancer.
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= Breast
= Lung
= Ovarian
= Leukemia
BRCA1+
= Prostate
BRCA1‐
Breast Cancer Prevention
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The Redactome
Predispositional Testing Laura is now 60 years old. She has been in good health. She and her husband have heard about the possibility of having genomic testing, and explore the possibilities on the internet.
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Genome‐Wide Association Studies
Tag SNP
Recombination hot spot
Gene
Symbol
Interleukin
IL‐4, IL‐13
Cluster of differentiation
CD14
2‐Adrenergic receptor
B2AR
Human leukocyte antigen DRB1, DQB1
HLA‐DRB1, HLA‐DQB1
Tumor necrosis factor
TNF
High‐affinity IgE receptor
FCER1B
Interleukin‐4 receptor
IL4RA
Disintegrin and metalloproteinase domain 33
ADAM33
Bierbaum, S., Heinzmann, A. Resp Med doi:10.1016/j.rmed.2007.01.018
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The “Dark Matter” in the Genome
Manolio et al. Nature 461:747, 2009
Direct to Consumer Genomic Testing
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Pharmacogenetics excretion
7-hydroxywarfarin
CYP2C9
S-Warfarin
NADH
NAD+ VKORC1
Vitamin K oxidized
Vitamin K reduced
activated clotting factors
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Genomics and Epigenomics
Personalized Genomics
Chen R et al. Cell 2012;148:1293‐1307.
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Where Will Personal Genomes Live?
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Research & Clinical Care
4/19/2012
UAB Personalized Medicine
Genome Sequencing
Phamarcogenetics
Patient & Provider Education
NIH Undiagnosed Disease Program
St. Hilaire C et al. N Engl J Med 2011;364:432-442.
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Strategic Initiative Genomics & Proteomics The principal strategic goal in genomics and proteomics at UAB is to empower investigators, which means to provide support to experienced investigators and to help “bootstrap” less experienced investigators to build genomics and proteomics into their research.
Heflin Center for Genomic Sciences Director Bruce Korf
Genomics Core Molly Bray
Proteomics Jim Mobley
Analytic & Epidemiologic Genomics Hemant Tiwari
Sequencing
Bioinformatics
Mike Crowley
David Crossman
Personalized Medicine Bruce Korf
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Genomics Pipeline at UAB Sample Acquisition HEFLIN CENTER FOR GENOMIC SCIENCES • Clinics Low to High Throughput Genomic and • Hospital Genetic Assays • Offsite locations Focused Next Gen Sequencing and GX
Study Design and Consultation
Sample Acquisition • Clinics • Hospital • Offsite
Sample Processing • Logging and Storage • DNA/RNA extraction • Assay Preparation
Whole Genome and Custom Data Analysis • Sequence analysis • Sequencing • Gene expression • Genotyping • Methylation
• Gene expression analysis • Genotyping analysis • Methylation analysis
Sample Acquisition HUDSON ALPHA INSTITUTE • Clinics • Hospital Large Scale Next Gen and • Offsite locations Specialized Sequencing Projects, High Volume Genomics Assays
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Next Generation Sequencing Technology and Services at UAB • Illumina Sequencing Resources (in collaboration with the Stem Cell Institute): – Hi‐Seq 2000 and GAIIx Next Generation Sequencers – cBot and Covaris S2 for Illumina HiSeq2000 and GAIIx Next‐Gen library prep
Next Generation Sequencing at UAB • HiSeq2000 – The HiSeq2000 can produce ~600 billion bases (Gb) of sequence per run (300 billion per flowcell with 2 flowcells) – The GAIIx can produce up to 95 billion bases (Gb) of sequence per run • NGS Assays include: – mRNA‐Seq – ChIP‐Seq – Exome Sequencing – Whole Genome Sequencing – Targeted Re‐sequencing – Microbiome
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UAB Illumina Technology and Services • Illumina Array Resources – iScan ‐ High density and high throughput genotyping, expression and methylation arrays – BeadXpress ‐ Medium density and throughput genotyping and methylation assays – Tecan robotics for accurate and efficient liquid/sample handling
Illumina Microarrays Offered Chip HumanOmni5.0-4 HumanOmni-2.5-8 + 2.5S HumanOmni-1 Quad + 1S HumanOmni Express-12 HumanExome + OmniExpressExome Human Linkage-24 Human CytoSNP iSelect custom arrays High density genotyping arrays for cow, dog, maize, pig, and sheep Human HT-12 gene expression BeadChip
Content >4.3 million >2.5 million >1,000,000 (new design) 700K + up to 200K custom >250K ~6100 ~300K Custom up to 1 million ~50K-700K
~47 K curated and non-curated transcripts and ESTs ~45 K curated and non-curated transcripts Mouse WG6 gene expression BeadChip and ESTs Mouse Ref-8 gene expression BeadChip ~26 K curated transcripts and ESTs Human Methylation450 ~450K putative methylation sites
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Microarray vs. NGS: Cost Comparison Microarray
Next Generation Sequencing
Assay
Cost*
Assay
Cost*
Gene expression
$245-$650
mRNA-Seq
$620§
Methylation
$365
Methyl-Seq
$650-1800†
ChIP-Chip
$550
ChIP-Seq
$650-1800†
Genotyping
$80-$620
Whole exome
$1,250§
Whole genome
$5,000ª
*Prices include labor and consumables and are subject to change §Price reflects running 28 samples per flowcell on HiSeq2000 †Prices reflect running several samples per lane v. one sample per lane ªPrice is for running 3 genomes per flowcell
NGS analysis is always going to provide more comprehensive data than an off-the-shelf microarray.
Microarray vs. NGS: Sample Quality and Quantity Microarray
Next Generation Sequencing
Assay
Amount
Assay
Amount
Gene expression
~300 ng total RNA
mRNA-Seq
1µg of Total RNA
Methylation
500 ng DNA
Methyl-Seq
10ng* of ChIP’d DNA
ChIP arrays
ChIP’d DNA
ChIP-Seq
10ng* of ChIP’d DNA
Genotyping
200-400 ng DNA
Whole exome
2-3µg DNA
Whole genome
All assays work best with good quality, un-degraded sample.
1µg DNA *Minimum amount
Illumina has developed “rescue” reagents for FFPE samples that can greatly increase yield.
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Data Analysis “Road Map” • Several groups on campus with capabilities to analyze NGS data: • • • • •
Heflin Genomics Core – David Crossman, PhD Statistical Genetics – Hemant Tiwari, PhD Pathology Group – Jonas Almeida, PhD CCTS – Elliot Lefkowitz, PhD CCC Statistics Core
• Creation of an analysis “clearing house” web page • General information on data complexity • Mechanism for triaging workflow and balancing work queues • Links to sites for analysis
UAB Program in Genomics & Proteomics Genomics & Proteomics Study Navigator • • • •
Consultation service Inventory Needs assessment Genomics & proteomics roadmap • Business case for master’s level study consultants Heflin Center/CCTS
Michelle Amaral, PhD
www.heflingenetics.uab.edu
Genomics & Proteomics Educational Coordinator • Education & outreach program • Innovative educational offerings
HudsonAlpha N. Susan Huffman, PhD
[email protected]
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Amara’s Law
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