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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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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13 14 15 16 17 1819 20 21 22

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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

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irs

150

200

250

300

$10, 000

$1, 000

$100

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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

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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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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

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ASSEMBLY

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Dilution series shows severity of yeast mutant respiratory growth defects

35°C

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atp6-T8993C

atp6-T9176G

atp6-T9176C

atp6-T8851C

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% ATP synthesis

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8

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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

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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

Chemical genomics in yeastExploiting yeast genome-wide collections

Hoon et al., Nat Chem Biol 2008

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

~1 Day Ago

February 2011NHGRI Published New Vision for Genomics