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Chapter 15
Genomics and Medicine
The impact of genomics on the practice
of medicine
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Contents




Medical promise of genomics
High-throughput methods for genotyping
Cancer genomics
Microbial genomics and medicine
 Finding new drug targets
 Developing vaccines
 DNA vaccines
 Gene therapy
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
What we hope to gain from genomics
 Drug, diagnostics, and prognostics development
 Genotyping to predict patient susceptibility to disease
 Personalized healthcare based on an individual’s
genomic features
genome
decision support systems
genotype
health
molecular profile
patient history
knowledge base
drugs diagnostics prognostics
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Long term returns
 Personalized genotype databases
 Used to assess health risks throughout life
 Adjustments to lifestyle and medical treatment
 Simulated cells
 Reduce the need for time consuming
experiments
 Allow experiments that would otherwise be
impossible
 New frameworks for clinical trials
 Pharmacogenomics
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Short term returns
 Faster characterization of disease genes
 Better disease diagnosis / prognosis with
microarrays
 Better methods for genotyping
 More efficient drug / vaccine development
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Advances in disease genetics
250
Disease genes
 Detection of disease
genes is most direct
medical use of genomics
information
 Over 1,000 disease
genes were
characterized by 2000
 How to exploit this
information?
0
Year of discovery
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Preimplantation diagnosis
 Couples with at least one child suffering from cystic
fibrosis underwent preimplantation diagnosis
 Biopsied cells from in vitro 3-day old embryos were
genotyped
 Implanted embryos (NN and ND) in one couple resulted in
a healthy baby girl
1
2
N D N D
3
4
5
N D
N D
N D
6
N D
biopsied cell
DNA added
heteroduplex
homoduplex
DD
NN
BAD
ND
NN
DD
diagnosis
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Single nucleotide polymorphisms (SNPs)
 Benefits of characterizing SNPs
 High density SNP map will greatly facilitate finding
disease genes
 Detection of SNPs can serve as a diagnostic for genetic
diseases
 Millions of SNPs presently in public and private
databases
 Fast, cheap, and accurate genotyping of SNPs still a
challenge
 Smallest linkage disequilibrium studies still out of
reach
 Genotyping 30,000 SNPs in 1,000 individuals required
10-fold increase in technological capacity at end of
2001
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Biochemical basis for SNP genotyping
 Hybridization with allele-specific
oligonucleotides (ASOs)
 Allele-specific primer extension
 Minisequencing
 Oligonucleotide ligation
 Restriction site cleavage
 Invasive cleavage
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Hybridization of allele-specific
oligonucleotides
 Uses hybridization to
detect SNP
 Heat or electric field
used to denature hybrids
 Caveat
 Each SNP hybrid will
denature with different
parameters
 Microarrays overcome
this to some degree
Allelle-specific
probes
Stable
Unstable
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Allele-specific primer extension
 Use allele specific
primers that include the
SNP
 PCR will extend primers
that match SNP, but not
mismatches
 Detection
 Fluorescence assay that
detects incorporation
of primer into PCR
product
Allele-specific
primers
PCR
Extension
No extension
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Minisequencing
 Use DNA polymerase to
extend primer sequence
by a single nucleotide
 Detection
 Colorimetric assay
using antibodies to
chemical group
attached to nucleotide
 Luminometric
detection of chemical
released upon
nucleotide addition
 Mass spectroscopy
Minisequencing
primer
PCR
One-nucleotide
extension
No extension
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Oligonucleotide ligation
 Three probes used
 Increases cost
 Ligase ties together
matched probes
 Detection methods
 Changes in mobility
 Microarrays
Left probes
Right probe
Ligase
Match,
ligation
Mismatch,
no ligation
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Restriction site cleavage
 Presence of SNP creates
a site for cleavage by
restriction enzyme
 Pattern of restriction
fragments reveals
presence (or absence) of
SNP
 Method not sufficient
for genome-wide SNP
scan
Restriction enzyme
Cleavage
No cleavage
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Invasive cleavage
 Highly specific probe
binds to target sequence
 Probe causes change in
conformation of double
stranded oligonucleotide
 New conformation
provides target for
cleavage by FLAP
endonuclease
 Cleaved signal sequence
indicates SNP
 Requires a lot of DNA
Invader probe
Flap
Endonuclease
Match,
no cleavage
Mismatch,
cleavage
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
An ideal SNP genotyping method
 PCR is rate limiting step
of most SNP genotyping
techniques
 The ideal genotyping
method
 Single molecule
genotyping (i.e. no
PCR)
 Example: atomic force
microscopy with
nanotube probes
AFM
probe
DNA
labels
DNA
molecule
height
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Cancer genome projects
 Cancer Genome Anatomy Project (CGAP)
 Established 1997 by National Cancer Institute (USA)
 Specializes in EST sequencing
 Human Cancer Genome Project (HCGP)
 Established 1999 by Brazilian research groups
 Specializes in SAGE analysis
 Cancer Genome Project (CGP)
 Established 2000 by Wellcome Trust and Sanger
Institute (United Kingdom)
 Specializes in genomic mutations leading to cancer
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Cancer genomics using ESTs and SAGE
 Use EST/SAGE tags from normal and tumorous tissue
 Tags stored in publicly accessible databases
 Bioinformatics tools used to reveal patterns of gene
expression that define cancerous states
database and
analysis
normal
SAGE/EST
sequencing
tags
tumor
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Searching for cancer-causing
mutations in genomic DNA
 Use human genome
sequence to make PCR
primers for target genes
 Compare PCR products
from normal tissue and
tumors using automated
heteroduplex analysis
 Sequence genes when
heteroduplex analysis
suggests tumor/normal
differences
 Find genomic mutants
Make PCR primers of
target genes from normal
and tumor tissue
1
PCR
2
heteroduplex analysis
3
sequence mutants
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Benefits of cancer genomics
 EST/SAGE projects
 Annotations for human genome sequence
 Understanding cancerous / normal tissue
differences in gene expression
 Identifying cancer-specific splice variants
 Genetic polymorphisms associated with cancer
 Investigation of genomic DNA
 Genetic polymorphisms associated with cancer
 Identification of cancer-causing mutations
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Microarrays and cancer
 Histology not always effective tool for
prognosis/diagnosis
 Microarrays distinguish cancerous tissues on
the basis of a gene expression profile
 Use in diagnosis
 Example: characterizing acute lymphoblastic
leukemia
 Use in prognosis
 Example: metastasis in medulloblastoma
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Microarrays in the prognosis of
metastasis
 Identified 85 genes with
different levels of
expression in metastatic
and non-metastatic
tumors
 72% accuracy in
predicting metastasis
 Identified genes that
induce metastasis
M–
M+
Downregulated
Up-regulated
 Could serve as
potential drug targets
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Microbial genomics and medicine
 Hundreds of microbial genomes have been
sequenced
 Opportunities for better understanding disease
 Reveal new drug targets
 Suggest vaccine candidates
 Most microbial genomes sequenced are
pathogenic
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Malaria
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Infects 500 million, kills ~2 million every year
Mosquito-borne illness
Drug and pesticide resistance emerged in the 1960’s
Global warming may increase size of endemic areas
1994
1966
1946
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The life cycle of P. falciparum
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Advances in genomics and malaria
 2003: human genome
sequence completed
 2002: Complete genome
of mosquito
 2002: Complete genome
of P. falciparum
 Genomic approaches to
combating malaria
 Genetically modified
mosquitoes
 Efficient development
of drugs / vaccines
genetically modified
mosquitos
P. falciparum
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Using transgenic mosquitoes to
control malaria
 12-amino acid peptide (SM1) found to inhibit
Plasmodium entry to salivary glands
 Transgene used to transfect germline of mosquitoes
 CP promoter for gene expression during blood feeding
 GFP to detect transfection
 4 copies of 12-amino acid peptide gene
CP signal
GFP
AgCP
promoter
HA1
[SM1]4
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Ability of transgenic mosquitoes to
infect was impaired
 69-95% decrease in
oocyst formation
 Infection of mice greatly
reduced or eliminated
 Caveats
 P. falciparum can
evolve to overcome
transgenic mosquitoes
 Need more transgenes
to reduce this
possibility
GM
midgut
Wild-type
midgut
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Using functional genomics to find drug
targets
 Functional genomics reduces the need for
complex biochemical analysis
 Genome sequence sufficient to reveal
undiscovered pathways
 Functional genomics can identify previously
characterized proteins in a new species
 In some cases providing targets for pre-existing
drugs
 Example: P. falciparum
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Bacterial homologs in P. falciparum
 The apicoplast: an essential organelle in P. falciparum
 Self-replicating
 Possesses its own 35 kb genome
 Related to algae
 Using sequences from the P. falciparum genome
project, a bacterial enzyme was discovered
 DOXP reductoisomerase, part of synthetic pathway
 Homologs in E. coli, B. subtilis, and Synechocystis sp.
 Enzyme believed to be associated with apicoplast
Pfal
Ecol
Bsub
Syne
LDNNKVLKTKCLQDNFSKINNINKIFLCSSGGPFQNLTMDELKNVTSENALKHPKWKMGKKITIDSATMMNKGLEVIETH
LPQPIQHNLGYADLE---QNGVVSILLTGSGGPFRETPLRDLATMTPDQACRHPNWSMGRKISVDSATMMNKGLEYIEAR
LQ----------GEQ---AKNIERLIITASGGSFRDKTREELESVTVEDALKHPNWSMGAKITIDSATMMNKGLEVIEAH
LQ----------GVP---EGGLRRIILTASGGAFRDLPVERLPFVTVQDALKHPNWSMGQKITIDSATMMNKGLEVIEAH
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The mevalonate and DOXP pathways
 Synthesis of isopentenyl
diphosphate
 Essential for synthesis
of steroids
 DOXP pathway used
by bacteria and plants
(chloroplasts)
 Mevalonate pathway
used by animals, fungi
 Drug targeting DOXP
pathway should have
few human side-effects
OH
OH
HOOC
CO-SCoA
OP
O
OH
DOXP
OH
HOOC
OH
HO
Mevalonate
OP
O
OH
HO
OH
OP
HOOC
OPP
OH
OH
OPP
isopentenyl diphosphate
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
 GFP-labeling showed
isolation of DOXP
redectoisomerase in
apicoplasts
 Previously developed
antibacterial drug FR900098 effectively
inhibits DOXP
reductoisomerase
 FR-900098 cured rats
with Malaria with
minimal toxicity
enzyme activity (%)
Can drugs that target DOXP
reductoisomerase cure malaria?
100
80
60
40
20
0
0.1
1
10 100 1000
drug concentration (nM)
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Finding vaccine candidates
 Genomics for vaccine development
 Functional genomics reveals microbial surface
proteins
 Surface proteins constitute potential antigens
for use as vaccines
 Recombinant antigen proteins used to test new
vaccines
 Example: N. meningitidis
 32% of all meningococcal disease in U.S.
 Genome fully sequenced in 2000
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Challenges in N. meningitidis vaccine
development
 1960’s: Using purified membrane-associated
polysaccharides, a vaccine was developed
 Worked well on adults
 But ineffective in most vulnerable population
 Children and infants
 No vaccine for serogroup B N. meningitidis
 Serogroup B vaccines using polysaccharides
too similar to human polysaccharides
 Serogroup B vaccines using surface proteins
are too strain-specific
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Using genomics to overcome vaccine
challenges
 Identified 570 putative membrane proteins in
serogroup B Meningococcus (MenB)
 Express proteins in E. coli
 Look for positive immune responses in mice
 Select vaccine candidates expressed on the
surface of multiple virulent strains of MenB
 Narrow down vaccine candidates to 7 antigens
that lack phase variability
 Look for surface proteins in other Neisseria
species
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DNA vaccines
 Inject naked DNA
containing microbial
gene into patient
 Somatic (or preferably
antigen-presenting) cells
produce DNA product,
which constitutes
antigen
 Host generates immune
response to antigen
A gene gun
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Components of a DNA vaccine plasmid
 Origin of replication for
rapid replication in
bacteria
 Antibiotic resistance gene
to select transfected
bacteria
 Mammalian promoter
 PolyA tail for mRNA
stabilization
 CpG motif for strong
immune response
 Antigen gene
Origin
Antibiotic
resistance
Promoter
Gene
insert
Poly A tail
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DNA vaccines under development
 >600 DNA vaccines
currently under
development
 Examples of diseases
being tackled
Clinical Trials (2002)
 Tuberculosis
 Malaria
 AIDS
 Presently no DNA
vaccines on the market
I
I-II
II
II-III
III
phase
Total number
636
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Advantages and disadvantages
compared to traditional vaccines
 Advantages of DNA vaccines

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
Induce humoral and cellular immune responses
Manufactured very easily
Inexpensive
No refrigeration necessary
 Disadvantages
 Concerns about autoimmune disease
 Possibility of introducing foreign DNA into the
human germline
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Gene therapy
 Goal: Introduce a
working copy of a gene
into somatic cells where
gene function is lacking
 Example: 4 year old girl
treated for hereditary
immune disease using
transfusion of
transduced T-cells
Gene therapy in
monkey muscle cells
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Gene therapy vectors
 Viruses
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Adenovirus
Adenoviruses
Adeno-associated
Retroviruses
Good for long-term
therapy
 Naked DNA and
liposomes
 Good for short-term
therapy
 Weaker immune
response
capsid
protein
Therapeutic DNA
Liposome
lipid
bilayer
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Gene therapy success and failures
 Some successes
 12/20 volunteers cured of angina following
injection of angiogenic gene directly into heart
 A transgenic mouse with sickle cell anemia was
cured following injection of healthy gene using
an HIV-like virus
 Some tragic failures
 An 18 year old volunteer died from a massive
immune response following gene therapy
 Fatal leukemia?
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Gene therapy caveats
 Thus far most gene therapy experiments have
been successful in immune cells, muscle and
liver tissues
 Possibility of altering the human germline
 Dangerous immune responses to vectors
 Implication of retroviral vectors in causing
cancer
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Summary I
 Long-term promise of genomics in medicine
 Personalized genotype databases
 Simulated cells
 Revolution in drug development
 Short-term prospects




More rapid characterization of disease genes
Better methods for genotyping
Microarrays for diagnosis and prognosis
Database of cancer gene expression
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Summary II
 Short term prospects (continued)



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New microbial drug targets
New microbial vaccines
DNA vaccines
Gene therapy
© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458