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Genomics and The Future of Medicine Gene chip diagnoses and designing ourselves
T Mueller
Division of Nephrology and ImmunologyUniversity of Alberta
Technology and Future of Medicine CourseNov 02, 2011
Leonard Cohen and Medicine
Thomas F MuellerDivision of Nephrology and Immunology
University of Alberta
International Leonard Cohen FestivalWestin HotelJuly 24, 2008
Leonard Cohen Medicine
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leonardcohenforum.com • Search25 Jan 2008 ... Leonard Cohen, A Life In Art. I often wondered whether Leonard had ... and Dr. Thomas Mueller who will speak on "Leonard Cohen's Medicine". ... www.leonardcohenforum.com/search.php?keywords=nadel - 32k -
Leonard Cohen Medicine
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Topics1. Background
• Human Genome Project• Gene Expression
2. Techniques• PCR• Microarray• Sources• Analysis
3. Application • Strengths and weaknesses• Outcome
4. Summary and Outlook• OGOD• Integration of data• Clinical phenotyping
Initial sequencing and analysis of thehuman genome
International Human Genome Sequencing ConsortiumNature 2001, VOL 409, 15 FEBRUARY 2001
Science 16 February 2001: Vol. 291 no. 5507 pp. 1304-1351
The Sequence of the Human GenomeJ Craig Venter et al.
The Human Genome Project
• book of life• would strangle research• genome race between public and private• White House announcement June 2000• Wall Street promises of genetic crystal balls
The Human Genome Project
• book of life• would strangle research• genome race between public and private• White House announcement June 2000• Wall Street promises of genetic crystal balls
• cancer (most diseases) not yet cured• overall disappointment• …
How many genes are in the human genome?
Genome experts … not even close …Science 2007; 316: 1113
How many genes are in the human genome?
… about 20,000 protein-coding genes …
Science 2007; 316: 1113
The Human Genome Project
• greatest impact of genomics• to investigate biological phenomena in a
comprehensive, unbiased, hypothesis-free manner
• in basic biology understanding of • protein-coding genes• non-coding genes• regulatory sequences
• in medicine to discover• gene and pathways underlying diseases• about 2,850 genes underlying Mendelian diseases• about 1,100 loci affecting common polygenic disorders• and about 150 new recurrent targets of somatic mutation in cancer
• costs of DNA sequencing plummeted by around 100,000 fold• RNA-Seq is also being applied to RNA transcripts to count their abundance• sequencing will become a routine tool to characterize
• patient’s individual genomes• cancer genomes• immune repertoires, and• microbiomes
Where is molecular profiling today?
Where is molecular profiling today?
ease of implementation
bene
fit to
the
heal
th
piece of cakegoing to the moon would be easier
zilc
hra
dica
l
My personal experience and journey …
Where is molecular profiling today?
ease of implementation
bene
fit to
the
heal
th
piece of cakegoing to the moon would be easier
zilc
hra
dica
l
2004
2011
Naesens, Sarwal, Nat Rev Practice Nephro 2011
Naesens, Sarwal, Nat Rev Practice Nephro 2011
Topics1. Background
• Human Genome Project• Gene Expression
2. Techniques• PCR• Microarray• Sources• Analysis
3. Application • Strengths and weaknesses• Outcome
4. Summary and Outlook• OGOD• Integration of data• Clinical phenotyping
DNA synthesis(replication)
RNA synthesis(transcription)
protein synthesis(translation)
DNA
RNA
Protein
amino acids
reverse transcription
polymerase chain reaction
continuously monitored
real-time RT-PCR
PCR – an alternative to measure transcription
1 1
2 2
3 4
4 8
5 16
6 32
7 64
8 128
9 256
10 512
11 1024
12 2048
13 4096
14 8192
15 16384
16 32768
…
49 2.81475E+14
50 5.6295E+14
51 1.1259E+15
52 2.2518E+15
53 4.5036E+15
54 9.0072E+15
55 1.80144E+16
56 3.60288E+16
57 7.20576E+16
58 1.44115E+17
59 2.8823E+17
60 5.76461E+17
61 1.15292E+18
62 2.30584E+18
63 4.61169E+18
64 9.22337E+18
amplification
http://images.google.com/imgres?imgurl=http://www.sjgames.com/proteus/img/chessboard.jpg&imgrefurl=http://www.sjgames.com/proteus/&h=1210&w=1200&sz=198&tbnid=lezl536gQYMJ:&tbnh=149&tbnw=148&prev=/images?q=chessboard&hl=en&lr=&oi=imagesr&start=1
DNA synthesis(replication)
RNA synthesis(transcription)
protein synthesis(translation)
DNA
RNA
Protein
amino acids
cDNA synthesis
transcription and labelling
oligonucleotide microarrays
hybridization and staining
Principle of microarrays
Affymetrix® info material
Principle of microarrays
Affymetrix® info material
Principle of microarrays
Affymetrix® info material
Looking at the stars
Baby steps
Bittner, Meltzer, Trent. Nature Genetics 1999; 22: 213.
In the film What About Bob … Bill Murray plays a mental patient with a multi-phobic personality whose fear of almost everything leaves him in a constant state of panic. …
… an eminent psychiatrist whose therapy goals for Bob are summed up in his book entitled Baby Steps, in which he advocates setting small, reasonable goals one day a time – one tiny step at a time.
The multi-phobic character will resonate all too well with the geneticist who faces the rapid evolution of bioinformatics driven by massive quantities of data produced by genome-scale technologies. This technology brings us face to face with the underlying complexity of biological systems and genome-wide function … and the key to success is cross-disciplinary collaboration –and effective communication – with those who develop data analysis and integration tools - the mathematicians.
Data analysis and integration: of steps and arrows
Matrix of transcript levels
Signature
Profile
Gene 1Gene 2
.
.
.
Gene M
Sample 1 Sample 2 . . . Sample N
Visualization and analysis of transcript profiles by heatmaps, dendrograms, and clusters
Bittner, Meltzer, Trent. Nature Genetics 1999; 22: 213.
Bittner, Meltzer, Trent. Nature Genetics 1999; 22: 213.
Analysis of microarray transcript data
Topics1. Background
• Human Genome Project• Gene Expression
2. Techniques• PCR• Microarray• Sources• Analysis
3. Application • Strengths and weaknesses• Outcome
4. Summary and Outlook• OGOD• Integration of data• Clinical phenotyping
Living donorkidneys (n=76)
Deceased donor kidneys (n=67)
1hour
Kidney biopsy
Microarrayanalysis
recipient
Clinical data
Clinical data
analysis
1-2 days
GC project
Renal scan(DTPA or MAG3)
HE stained implant biopsies of living and deceased donors
C
A
ED
B
Which kidney is from a deceased donor?
C
A
ED
B
deceased deceasedliving donors
x
x
The transcriptome reflects donor origin
Mueller T et al, Am J Transplant 2008
Cluster 2:
DD Low riskCluster 3:
DD High riskCluster 1:
LD
The kidneys are clustered in relation to risk of delayed graft function (DGF)
x
x
Mate kidneys cluster together
Transcript levels of 10 acute phase response genes in implant biopsies
CEW OAL
Distribution of all individual kidneys based on their transcriptome changes
“good”“bad”
‘Outliers’ indicated by the transciptome changes
“good”“bad”
BP ↓↓
DGF +++
sepsis
kidney heart
The same transcripts are increased in Heart, Liver, and Kidney during Rejection
liver
d5/Co Gene
1075 Gzmb
827 Serpina3g
273 Ccl3
174 Ms4a4b
126 Slfn1
118 AI447904
111 Bcl2a1a
93 Lpxn
90 Cd3d
88 Socs1
d5/Co Gene
162 Gzmb
122 Cd3d
108 Serpina3g
92 Cd3g
75 Cd8b
73 AI447904
66 Ctsw
58 Ms4a4b
56 Ms4a4b
55 Lat
d10/Co Gene
50 Pglyrp1
29 Gng2
29 Emb
24 Cd3d
20 Myo1g
20 Pdcd1
17 Ly9
13 Cd8b
12 Cd37
12 Ltb
287
150common
Mean ↑ 19-fold Mean ↑ 6-fold Mean ↑ 38-fold
Mueller, Einecke et al, Am J Transplant 2008.
The continuity of changes in kidney biopsies
Where is molecular profiling today?
Do we have achieved personalized medicine?
“… we simply cannot accurately predict outcomes for the most clinically relevant sample size – a single patient.”
B Kaplan and J Schold
Odysseus or Hamlet – that is now the question
Science 2005; 309: 2010.
Differences in phenotypes in fingerprints of identical twins and the first cloned cat
Michiels S et alPrediction of cancer outcome with microarraysLancet 2005; 365: 488-92
“Findings: The list of genes identified as predictors of prognosis was highly unstable; molecular signatures strongly depended on the selection of patients in the training sets. … Because of inadequate validation … overoptimistic results … five of the seven studies did not classify patients better than chance.”
“Microarrays and molecular research: noise discovery?”
Systems biology approach
Naesens, Sarwal, Nat Rev Practice Nephro 2011
What was the discovery that made Galileo Galilei a tenured Professor
A discovery of 4 satellites resolving about Jupiter B contradicting that the earth was the centre of all celestial motionsC detecting approaching war ships 2 hours earlier D placing the Sun at the centre of the universeE describing the uniformity of acceleration independent of mass
Slide Number 1Slide Number 2Slide Number 3Slide Number 4Slide Number 5Slide Number 6Slide Number 7Slide Number 8Slide Number 9How many genes are in the human genome?How many genes are in the human genome?Slide Number 12Slide Number 13Slide Number 14Slide Number 15Slide Number 16Slide Number 17Slide Number 18Slide Number 19Slide Number 20Slide Number 21Slide Number 22Slide Number 23Slide Number 24Slide Number 25Slide Number 26Slide Number 27Slide Number 28Visualization and analysis of transcript profiles by heatmaps, dendrograms, and clustersSlide Number 30Slide Number 31Slide Number 32Slide Number 33Slide Number 34Slide Number 35The transcriptome reflects donor originThe kidneys are clustered in relation to risk of �delayed graft function (DGF)Mate kidneys cluster togetherSlide Number 39Slide Number 40Slide Number 41Slide Number 42Slide Number 43Slide Number 44Slide Number 45Slide Number 46Slide Number 47Slide Number 48Slide Number 49Slide Number 50Slide Number 51Slide Number 52Slide Number 53