Post on 13-Apr-2020
The road from genomics to personalized medicine
Raeka Aiyaraiyar@embl.deSteinmetz GroupGenome Biology UnitEMBL HeidelbergEUSJA Visit, 18 July 2011www.embl.de/research/units/genome_biology/steinmetz
EMBL
6 billion base pairs, 22K protein-coding genes, ~300K proteins
~6 million differences between two individuals
Green, ED et al. Charting a course for genomic medicine from base pairs to bedside. Nature (2011)
Understanding the structure of
genomes
Understanding the biology of
genomes
Understanding the biology of
disease
Advancing the science of
medicine
Improving the effectiveness of
healthcare
1990-2003Human Genome
Project
2004-2010
2011-2020
Beyond 2020
Personalized/P4 Medicine
• Predictive - development of probabilistic health projection based on individual DNA and gene expression
• Preventative - creation of therapeutics that will prevent a disease a person is at risk of developing
• Personalized - treating an individual based on their unique human genetic variation, complementing the predictive and preventative efforts above
• Participatory - patient's active, informed involvement in their medical choices and care, acting in partnership with their health providers
Watson & Crickdescribe the
DNA double helix
Draft human genome sequence
200!
Mendel discoverslaws of genetics
!865
!953
Nirenberg,Khorana &
Holley determinethe genetic code
!966
End of the Human Genome Project
2003
Mouse genomesequence
2002
Fruit!y (Drosophila melanogaster) genome sequence
2000
GenBank database established
!982
Yeast (Saccharomyces cerevisiae)
genome sequence
!996
Escherichia coli genomesequence
!997
Roundworm (Caenorhabditis elegans) genome sequence
!998
Human Genome Project launched
!990
!977
Sanger and Maxam & Gilbertdevelop DNAsequencing methods
G A T C
Publication of "nishedhuman genome sequence
Rat genomesequence
Dog genome sequence
Chimpanzee genomesequence
ENCODE pilot project complete
Honeybee genome sequence
Human genetic variation is breakthrough of the year
Platypus genome sequence
Comprehensive genomicanalysis of glioblastoma
First cancer genome sequence (AML)
Genetic InformationNondiscrimination Act (GINA)
passed in US
Bovine genome sequence
First human methylome map
Completion of the Mammalian Gene Collection (MGC)
500th genome-wide association studypublished
Wellcome Trust Case ControlConsortium publication
First direct-to-consumer whole-genome test
Korean genome sequence
International data release workshop
Southern African genome sequences
Neanderthal genome sequence
UK Biobank reaches500,000 participants
1000 Genomes pilot projectcomplete
Nu#eld Council on Bioethics publication on personalized healthcare
>1,000 mouseknockout mutations
modENCODE publications
Han Chinesegenome sequence
Yoruba genomesequence
Genomic achievements since the Human Genome Project
Sea urchingenome sequence
Publication ofhuman genome
2004Dog genom
2005wh
2006ge
2007Nondiscrim
pa
2008 Korean
2009 20!0
Miller syndrome genediscovered by exome sequencing
First genome-wide association study
published
1
Age-related maculardegeneration
CFH
First personal genome sequenced
1 2 3 4 5 6 7 8 9
Coding
UTR
c.56G
>A
c.403
C>T
c.454
G>A
c.595
C>T
c.605
A/C
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C>T
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6C>T
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5A>G
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Chicken genomesequence
Phase I HapMap
Rhesus macaquegenome sequence
NCBI's Database of Genotypes and Phenotypes (dbGaP) launched
First personal genomesequenced using new technologies
Moore’s law
For details, see http://genome.gov/sequencingcosts2002 2004 2006 2008 2010Cost per human genome sequence
Design by Darryl Leja (NHGRI, NIH).Watson and Crick photograph: A. Barrington Brown/Photo Researchers; images of Science covers courtesy of AAAS.
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© 2011 Macmillan Publishers Limited. All rights reserved
The success of genomics
Green, ED et al. Charting a course for genomic medicine from base pairs to bedside. Nature (2011)
The evolution of sequencing technology
Trac
e The TrTrT ace archive, started in 2000, houses raw sequence data, and currently holds 1.8 trillion base pairs.
chiw sequenw sequen
ently holds 1.8 trillion base paiently holds 1.8 trillion base pai s.
20012000 2002 2003 2004 2005 2006 2007 2008 2009 2010
50
0
100
Billi
ons o
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e pa
irs
150
200
250
300
$10, 000
$1, 000
$100
$10
$1
Cost per million base pairs of sequence ( log scale)
AUTOMATED SANGER SEQUENCING: Based on a decades-old method, at the peak of the technique, a single machine could produce hundreds of thousands of base pairs in a single run.
SEQUENCING BY LIGATION:This technique employed in SOLiD and
chemistry from previous technologies and samples every base twice, reducing the error rate.
454 PYROSEQUENCING :Released in 2005, 454 sequencing is considered the first ‘next-generation’ technique. A machine could sequence hundreds of millions of base pairs in a single run.
SEQUENCING BY SYNTHESIS:Other companies such as Solexa (now Illumina) modified the next-generation, sequencing-by-synthesis techniques and can produce billions of base pairs in a single run.
Whole Genome Shotgun Sequence
Gene sequence stored in
international public databases
THIRD-GENERATION SEQUENCING: Companies such as Helicos BioSciences already read sequence from short, single DNA molecules. Others, such as Pacific Biosciences, Oxford Nanopore and Ion Torrent say they can read from longer molecules as they pass through a pore.
Human genome at ten: The sequence explosion. Nature (2010)
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Metagenomic& heterogeneous
samples
Compoundlibraries
Environmentalexposures
Tissue types& substructures
Transcriptionallyactive sites
Protein-DNAinteractions
mRNAexpression& discovery
microRNAexpression& discovery
Alternativesplicing
& allele-specific expression
DNA bar codes
Mutationdiscovery& profiling
Copy numbervariation
PopulationsTemporalchanges
Kahvejian A et al., Nature Biotechnology (2008)
What would you do if you could sequence everything?
Complex diseases
• Do not follow Mendelian inheritance and result from multiple alleles and environment
• Responsible alleles contribute different amounts to phenotype
• Alleles may be present in only a fraction of all individuals with the phenotype
• Need large sample sizes and high density marker maps to find alleles
Peltonen, L & McKusick, V. SCIENCE Online 2001
Gingeras, TR. Origin of phenotypes: genes and transcripts. Genome Research (2007)
• 70% of human/mammalian genomes(Carninci et al. 2005)
• 85% of yeast genome(David et al. 2006)
Pervasive transcription covers:
Most of the genome is transcribedUnderstanding the biology of
genomes
Zhenyu Xu
Wu Wei
Julien Gagneur
Sandra Clauder-Münster
Bidirectional promoters generate pervasive transcription
• >30% of promoters are bidirectional, accounting for >50% of unannotated transcripts in the genome
Xu, Z., Wei, W., Gagneur, J., Perocchi, F., Clauder-Muenster, S., Camblong, J., Guffanti, E., Stutz, F., Huber, W. and Steinmetz, L.M. Bidirectional promoters generate pervasive transcription in yeast. Nature 2009.
Neil, H., Malabat, C., d’Aubenton-Carafa, Y., Xu, Z., Steinmetz, L.M. and Jacquier, A. Widespread bidirectional promoters are the major source of cryptic unstable transcripts in yeast. Nature 2009.
d1
d2
Promoter bidirectionality is universal
–2 kb
–2 kb
–1 kb
–1 kb
CUTs and SUTs
Promoter region
PALRs
Non-capped: TSSa-RNAs
Capped:
NRO-RNAs
PROMPTs
mRNA
mRNA
DNA
a Yeast
b Mammals
Long
RN
AsLo
ng R
NAs
Shor
t RN
As
Gene
Gene
PASRs
Yeast:Xu, Z. et al., Bidirectional promoters generate pervasive transcription in yeast. Nature Jan. 2009
Neil, H. et al., Widespread bidirectional promoters are the major source of cryptic transcripts in yeast. Nature Jan. 2009
Human: Leighton, J. et al., Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science Dec.
2008
Preker, P. et al., RNA exosome depletion reveals transcription upstream of active human promoters. Science Dec. 2008
He, Y. et al., The antisense transcriptomes of human cells. Science Dec. 2008
Mouse: Seila, A.C. et al., Divergent transcription from active promoters. Science Dec. 2008
Parasites:Teodorovic et al., Bidirectional transcription is an inherent feature of Giardia lamblia promoters and contributes to an abundance of
sterile antisense transcripts throughout the genome. Nucleic Acids Res. 2007
Carninci, Nature (2009)
Regulatory roles of antisense expression
• Transcription interference:
• IME4 (Hongay et al., 2006)
• Inactivating histone modification:
• Pho84 (Camblong et al., 2007)
• Ty1 (Berretta et al., 2008)
• Gal1-10 (Houseley et al., 2008)
• Activating:
• Pho5 (Uhler et al., 2007)
ORFs with antisense are more often ‘switched off’
18% of ORFs with antisense are switched off vs. 8% without
0 4 6
0.00
0.10
0.30
Minimal expression levels across segregants
Den
sity
ORFs without antisenseORFs with antisense
Xu Z et al. Antisense expression increases gene expression variability and locus interdependency. Mol Sys Biol (2011)
Ultrasensitivity model for antisense function
Functional classes of genes with antisense transcripts:
cell fate decision genes (e.g. IME4)
condition-specific genes: plasma membrane and stress response genes
regulatory signal -> sense promoter -> sense activation
On state
TF
antisense sense expression
Off state
Non-coding transcription: Summary
• Pervasive transcription originates from bidirectional promoters
• Antisense transcripts in yeast play a regulatory role in switching genes off
• Increase sensitivity to genetic and environmental changes
• Beneficial for adaptation: greater response to different environments, cell-to-cell variation
Understanding the biology of
genomes
TF
Why are some mosquitoes resistant to the malaria parasite?
Which genetic factors determine resistance to Plasmodium?
Understanding the biology of
disease
Stephanie Blandin
Rui Wang-Sattler
Mapping mosquito QTLs for parasite resistance(L3-5 x 4Arr crosses)
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Blandin, S et al. Dissecting the genetic basis of resistance to malaria parasites in Anopheles gambiae. Science (2009)
TEP1 polymorphism confers resistance to P. berghei
Reciprocal allele-specific RNAi (rasRNAi)
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In progress: search for additional genetic factors
Blandin, S et al. Dissecting the genetic basis of resistance to malaria parasites in Anopheles gambiae. Science (2009)
Malaria and rasRNAi: Future Work
rasRNAi in human cell linesHeLa cells for mitosis phenotypesPatient derived lines
Field studies for malaria infectionMetagenomic sequencing to test association of mosquito and malaria genotypes in the field
Collaboration with Isabelle Morlais in Yaounde, Cameroon
Understanding the biology of
disease
Mitochondria: a diverse, essential organelle
Advancing the science of medicine
1000 proteins in yeast1500 proteins in human
ConservationMito: 60%Cell: 46%
Dual genetic originTightly integrated with cellular function
matrix innermembrane
space outermembrane
intermembranecristae
Yeast models of mitochondrial ATP synthase disorders
Cytosol
OM
IMS
IM
Matrix
Nucleus
18 subunits
Atp6p, Atp8p, Atp9p
18 subunits
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ASSEMBLY
Lefebvre-Legendre et al., JBC 2001
Dilution series shows severity of yeast mutant respiratory growth defects
35°C
Glucose(Fermentation)
atp6-T8993G
atp6-T8993C
atp6-T9176G
atp6-T9176C
atp6-T8851C
Wild type
fmc1Δ
% ATP synthesis
100
74
<5
6
8
7
50
Glycerol(Respiration)
35°C
Elodie Couplan, INSERM Brest
Components and structure of yeast mitochondrial ATP synthase
Severity of homologous mutations in yeast correlates with patient
phenotypes
Raeka Aiyar
Screen for chemicals active against ATP synthase disorders
10 11
Drug C7
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D
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g co
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tratio
n
Distance from the disk
Diffusion of the drugaround the disk
YEAST NARP
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~20 hits
Simple
Sensitive
Large dynamic range for dosage
Couplan E, Aiyar RS et al. A yeast-based assay identifies drugs active against human mitochondrial disorders. PNAS (2011)
High-throughput
Chemical genomics in yeastFinding drug targets via gene dosage
Gro
wth
Normal Inhibited by drug
Haploinsufficiency
Multicopy suppression
Deletionsensitivity
Overexpression
drug
Green, ED et al. Charting a course for genomic medicine from base pairs to bedside. Nature (2011)
Understanding the structure of
genomes
Understanding the biology of
genomes
Understanding the biology of
disease
Advancing the science of
medicine
Improving the effectiveness of
healthcare
1990-2003Human Genome
Project
2004-2010
2011-2020
Beyond 2020