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Preda Van Je 7 Dna Micro Array
Preda Van Je 7 Dna Micro Array
• DNA Replication
--ACGCGA--
--TGCGCT--
• RNA Transcription
--UGCGCU--
• Protein Translation
--CYSALA--
DNA
replication
transcription translation
5’ … TACTGAA … 3’
3’ … ATGACTT … 5’
Hydrogen Bond Makes DNA Binding Specifically
Hydrogen bond
5’
3’
5’
3’
Hydrogen Bond Makes DNA Binding Specifically
replication
transcription translation
(http://www.nhgri.nih.gov/)
Reverse Transcription
replication
transcription translation
Reverse Transcription
Cell A Cell B
Labeled cDNA
from geneX
Hybridizaton to chip
17
Oligonucleotide Arrays
• GeneChip® by Affymetrix
• Parallel synthesis of
oligonucleotide probes (25-
mer) on a slide using
photolithographic methods
• Millions of probes /
microarray
• Multiple probes per gene
• One-color arrays
18
Affymetrix GeneChip Probes
19
Labeled Samples Hybridize to DNA Probes on
GeneChip
20
Shining Laser Light Causes
Tagged Fragments to Glow
21
Perfect Match (PM) vs MisMatch (MM)
(control for cross hybridization)
22
Affymetrix Microarray Imagine Analysis
23
What is DNA Microarray?
• Scientists used to be able to perform genetic analyses of a few
genes at once. DNA microarray allows us to analyze thousands of
genes in one experiment!
Purposes.
• So why do we use DNA microarray?
– To measure changes in gene expression levels – two samples’ gene expression can be
compared from different samples, such as from cells of different stages of mitosis.
– To observe genomic gains and losses. Microarray Comparative Genomic Hybridization
(CGH)
– To observe mutations in DNA.
The Plate.
• Usually made commercially.
• Made of glass, silicon, or nylon.
• Each plate contains thousands of spots, and each spot contains a probe
for a different gene.
• A probe can be a cDNA fragment or a synthetic oligonucleotide, such as
BAC (bacterial artificial chromosome set).
• Probes can either be attached by robotic means, where a needle applies
the cDNA to the plate, or by a method similar to making silicon chips for
computers. The latter is called a Gene Chip.
Let’s perform a microarray!
1) Collect Samples.
2) Isolate mRNA.
3) Create Labelled DNA.
4) Hybridization.
5) Microarray Scanner.
6) Analyze Data.
STEP 1: Collect Samples.
This can be from a variety of organisms. We’ll use two
samples – cancerous human skin tissue & healthy human
skin tissue
STEP 2: Isolate mRNA.
• Extract the RNA from the samples. Using either a column, or a solvent
such as phenol-chloroform.
• After isolating the RNA, we need to isolate the mRNA from the rRNA and
tRNA. mRNA has a poly-A tail, so we can use a column containing beads
with poly-T tails to bind the mRNA.
• Rinse with buffer to release the mRNA from the beads. The buffer
disrupts the pH, disrupting the hybrid bonds.
STEP 3: Create Labelled DNA.
Add a labelling mix to the
RNA. The labelling mix
contains poly-T (oligo dT)
primers, reverse transcriptase
(to make cDNA), and
fluorescently dyed
nucleotides.
• The plates are convenient to work with because they are small.
• Microarrays: Chipping Away at the Mysteries of Science and Medicine. National Center for
Biotechnology Information, 27 July 2007. 19 Feb. 2010.
<http://www.ncbi.nlm.nih.gov/About/primer/microarrays.html>
• Brown, P.O. & Botstein, D. Exploring the New World of the Genome with DNA Microarrays. Nature
Genetics Supplement. 21. (1999): 33-37.
<http://www.ctu.edu.vn/~dvxe/Bioinformatic/PDF%20Files/Volume21/ng0199supp_33.pdf>
• Simon, R., Radmacher, M.D., Dobbin, K., & McShane, L.M. Pitfalls in the Use of DNA Microarray Data for
Diagnostic and Prognostic Classification. Journal of the National Cancer Institute. 95. (2003): 14-18.
http://jnci.oxfordjournals.org/cgi/content/full/95/1/14
• Holloway, A.J., Van Laar, R.K., Tothill, R.W., & Bowtell, D.D.L. Options Available – From Start to Finish –
For Obtaining Data From DNA Microarrays II. Nature Genetics Supplement. 32. (2002): 482-489.
<http://web.cs.mun.ca/~harold/Courses/Old/CS6754.W04/Diary/ng1030.pdf>
Presentation Overview
• Current application of pharmacogenomics in
drug development
• Industry concerns regarding the conduct of
pharmacogenomic studies and submission of
pharmacogenomic data
Current Applications of
Pharmacogenomics
• Primary uses relate to interpretation of clinical
trial results, data quality, study design, and
biomarkers.
• Targeting drugs at genetically-defined
populations is not a primary focus in
pharmaceutical development
• Three areas of greatest activity
– Clinical genotyping
– Pre-clinical gene expression
– Clinical gene expression
Clinical Pharmacogenetics
• Phase I studies
– Explain outliers or patient-to-patient variability
in PK
– Exclude or include specific patients
– Normalize genotype frequencies
– Bridge to other populations
Example: Desipramine PK Parameters
Genotyping can increase trial safety and explain outlying data
30
• Outlier has *6 null allele and *9
allele with reduced enzymatic
20 activity.
• Expected occurrence of null/*9
10 genotype is 0.4%
Katz et al., Abbott Labs.
Clinical Pharmacogenetics
• Phase II/III studies
– Identify genetically-defined groups with more
pronounced or rapidly progressing disease
– Exclude/include at-risk individuals
– Stratify studies based on genotypes
• Clinical response
• Risk of adverse events
– Where appropriate, develop drugs for specific
groups
– Identify genetic markers associated with clinical
outcomes
Example: Gene Association using SNP
Mapping
Association of anonymous SNP markers with Alzheimer’s Disease
25
APOe
Case-control p-value (-log10)
20
15
10
0
0 500 1000 1500 2000
-5
Distance (kb) Courtesy Allen Roses, GSK
Similar methods can be used to identify genes associated with drug effect
or drug adverse reactions
Pre-clinical Gene Expression
• Toxicogenomics
– Predict toxicity of candidate compounds
– Identify mechanisms of toxicity
• Identify potential biomarkers for toxicity or
efficacy for future clinical studies
Example: Toxicogenomics of
Hepatotoxic Compounds
52 compounds – rat liver mRNA analyzed by microarray
Enormous Amounts of
Homo sapiens cDNA: FLJ21197 fis, clone COL00201. 5.62E-06
rattus mRNA for transthyretin. 0.93
Mus musculus Ngfi-A binding protein 2 (Nab2), mRNA. 0.04
Homo sapiens solute carrier family 26, member 6 (SLC26A6), mRNA. 0.55
Rattus sp. pre-mtHSP70 mRNA, complete cds; nuclear gene for mitochondrial product. 0.58
EST225141 Normalized rat brain, Bento Soares Rattus sp. cDNA clone RBRCU93 3' end, mRNA sequence 0.96
Rat heme oxygenase-2 (HO2) mRNA, complete cds 0.3
Homo sapiens clone 23704 mRNA sequence. 0.09
EST106329 Rat PC-12 cells, untreated Rattus sp. cDNA clone RPCAZ49 3' 0.42
Rat mRNA for ribosomal protein S10 0.69
Homo sapiens mRNA; cDNA DKFZp434A1923 (from clone DKFZp434A1923); complete cds. 0.65
Data
Homo sapiens 5'-nucleotidase (purine), cytosolic type B (NT5B), mRNA. 0.84
us musculus B6D2F1 clone VI12H mRNA. 0.04
Rattus norvegicus small rec (srec) mRNA, complete cds. 2.49E-16
Homo sapiens cDNA: FLJ22539 fis, clone HRC13227. 0.45
Mus musculus, Similar to RIKEN cDNA 4930579A11 gene, clone MGC:7590, mRNA, complete cds. 0.99
Homo sapiens FK506 binding protein precursor (LOC51303), mRNA. 0.00252
Rattus norvegicus P450 (cytochrome) oxidoreductase (Por), mRNA. 3.51E-05
for interpretation
Rattus norvegicus cerebellar Ca-binding protein, spot 35 protein (Calb1), mRNA. 0.99
Rat mRNA for ribosomal phosphoprotein P2. 0.01
Homo sapiens partial mRNA; ID ED66-6B. 0.77
Homo sapiens hypothetical protein MGC10911 (MGC10911), mRNA. 4.77E-05
Mus musculus brain cDNA, clone MNCb-2609, similar to Homo sapiens mRNA; cDNA DKFZp434B027 (from clone 1.43E-12
DKFZp434B027).
Homo sapiens cDNA FLJ13323 fis, clone OVARC1001731, highly similar to TROPOMYOSIN ALPHA CHAIN, FIBROBLAST 0.31 ISOFORM F2.
Mus musculus, Similar to DKFZP566O084 protein, clone MGC:6908, mRNA, complete cds. 0.00496
Cytochrome P450, subfamily IVB, polypeptide 1 0.00349
Rattus norvegicus Myosin of the dilute-myosin-V family (Myr6), mRNA. 0.89
Rattus norvegicus activating transcription factor ATF-4 (Atf4), mRNA. 4.03E-06
t mRNA for ribosomal phosphoprotein P2. 0.04
Mus musculus GTP binding protein 2 (Gtpbp2), mRNA. 0.9
Mus musculus solute carrier family 7 (cationic amino acid, transporter, y+ system), member 10 (Slc7a10), mRNA. 0.71
Alanine-glyoxylate aminotransferase (Serine-pyruvate aminotransferase) 0.000183
NP_036136 acyl-CoA thioesterase 1, cytosolic [Mus musculus] 0.00025
O6-methylguanine-DNA methyltranferase 0.39
t mRNA for s-adenosylmethionine synthetase. 1
Mus musculus cell death-inducing DNA fragmentation factor, alpha subunit-like effector A (Cidea), mRNA. 0.53
Rattus norvegicus macrophage migration inhibitory factor (Mif), mRNA. 0.00255
t c-jun oncogene mRNA for transcription factor AP-1. 0.0013
Mus musculus mRNA for PS1D protein. 0.84
Gene p-value
IF 0.00679
Mus musculus laminin alpha 5 chain (Lama5) mRNA, partial cds. 0.79
t c-fos mRNA. 0.9
Mus musculus, Similar to PP1201 protein, clone MGC:7955, mRNA, complete cds. 0.79
Rattus norvegicus cytochrome P450 3A9 mRNA, complete cds 0.00153
EST199603 Normalized rat embryo, Bento Soares Rattus sp. cDNA clone REMAB23 5' end, mRNA sequence 0.00637
Mus musculus, Similar to PP1201 protein, clone MGC:7955, mRNA, complete cds. 0.79 Mus musculus pyruvate dehydrogenase kinase 4 (Pdk4), mRNA.
Cytochrome P450, subfamily IVB, polypeptide 1
Rattus norvegicus regulated endocrine-specific protein 18 (Resp18), mRNA.
arginase 1, liver
2.97E-12
1.12E-06
0.1
0.00118
Human mRNA for KIAA0182 gene, partial cds. 0.06
Rattus norvegicus cytochrome P450 3A9 mRNA, complete cds 0.00153 Mus musculus interferon-related developmental regulator 2 (Ifrd2), mRNA.
choline transporter
Rattus norvegicus D site albumin promoter binding protein (Dbp), mRNA.
Rattus norvegicus P450 (cytochrome) oxidoreductase (Por), mRNA.
0.17
0.03
0.000112
9.54E-12
EST199603 Normalized rat embryo, Bento Soares Rattus sp. cDNA clone REMAB23 5' end,
0.00637
mRNA sequence
at mitochondrial succinyl-CoA synthetase alpha subunit (cytoplasmic precursor) mRNA, complete cds. 0.09
SHGC-101755 Human Homo sapiens STS genomic, sequence tagged site. 0.06
Homo sapiens basic helix-loop-helix domain containing, class B, 2 (BHLHB2), mRNA. 1.91E-09
O6-methylguanine-DNA methyltranferase 0.12
Rattus norvegicus GTPase Rab6 mRNA, partial cds. 0.46
Homo sapiens hypothetical protein FLJ13448 (FLJ13448), mRNA. 0.74 Homo sapiens transmembrane protein induced by tumor necrosis factor alpha (TMPIT), mRNA.
Rat ARSB mRNA for arylsulfatase B, partial cds.
Mus musculus A kinase (PRKA) anchor protein (gravin) 12 (Akap12), mRNA.
icotinic receptor alpha 7 subunit [rats, brain, mRNA, 3030 nt].
0.76
0.36
0.66
0.25
Homo sapiens sorting nexin 10 (SNX10), mRNA. 1.01E-12
Rattus norvegicus DD6A4-1 mRNA, partial sequence. 0.95
Mus musculus RIKEN cDNA 3110023E09 gene (3110023E09Rik), mRNA. 0.000174
Homo sapiens chromosome 8 open reading frame 4 (C8ORF4), mRNA. 0.000963
Mus musculus RIKEN cDNA 0610040B21 gene (0610040B21Rik), mRNA. 0.79
Sequence Description Drug 1 P-value
Rattus sp. mRNA for kinase, complete cds. 1.14E-28
Mus musculus probable nocturnin protein mRNA, partial cds. 0.37
EST197805 Normalized rat placenta, Bento Soares Rattus sp. cDNA clone RPLAO46 3' 0.28
Lactate dehydrogenease B 0.00908
Mus musculus RIKEN cDNA 3222401G21 gene (3222401G21Rik), mRNA. 0.87
Mus musculus nicotinamide N-methyltransferase (Nnmt), mRNA. 0.09
Rattus norvegicus 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (Hmgcr), mRNA. 0.95
Rattus norvegicus p8 mRNA, complete cds. 0.01
GSTA5=glutathione S-transferase Yc2 subunit [rats, Morris hepatoma cell line, mRNA, 1274 nt]. 1.40E-22
Activating transcription factor 3 0.18
troponin I, cardiac 0.15
EST225890 Normalized rat brain, Bento Soares Rattus sp. cDNA clone RBRDF30 3' end, mRNA sequence 0.00481
B-cell translocation gene 2, anti-proliferative 0.02
us musculus putative serine 0.00163
Rattus norvegicus 3-phosphoglycerate dehydrogenase (Phgdh), mRNA. 0.09
Homo sapiens cDNA: FLJ21621 fis, clone COL07851, highly similar to HSU60061 Human FEZ2 mRNA. 0.05
Rattus norvegicus unconventional myosin Myr2 I heavy chain (Myr2), mRNA. 0.63
Mus musculus, Similar to hypothetical protein, clone MGC:6794, mRNA, complete cds. 0.22
Insulin-like growth factor binding protein 1 1.83E-05
at metallothionein-i (mt-1) mrna. 8.99E-10
Mus musculus, clone MGC:12123, mRNA, complete cds. 0.22
Homo sapiens, clone MGC:5487, mRNA, complete cds. 0.00334
Inhibitor of DNA binding 1, helix-loop-helix protein (splice variation) 0.56
Rattus norvegicus Apolipoprotein C-IV (Apoa4), mRNA. 1
T-kininogen 3.72E-14
Mus musculus RIKEN cDNA 4930586I02 gene (4930586I02Rik), mRNA. 0.39
Rattus norvegicus Activating transcription factor 3 (Atf3), mRNA. 0.61
Homo sapiens hypothetical protein FLJ13758 (FLJ13758), mRNA. 0.9
B-cell translocation gene 3 0.00858
Homo sapiens hypothetical protein FLJ13448 (FLJ13448), mRNA. 0.44
Mus musculus leucine rich repeat (in FLII) interacting protein 1 (Lrrfip1), mRNA. 0.33
Rattus norvegicus extracellular signal-regulated kinase 7 mRNA, complete cds. 0.000104
Mus musculus coenzyme A diphosphatase (Nudt7) mRNA, complete cds. 0.13
Rattus norvegicus glucocorticoid-induced leucine zipper (Gilz), mRNA. 5.65E-11
Mus musculus caspase 11 (Casp11), mRNA. 0.88
Homo sapiens hypothetical protein FLJ11565 (FLJ11565), mRNA. 0.97
Rattus norvegicus mRNA for LRp105, complete cds. 0.15
Rattus norvegicus glutathione S-transferase A3 subunit (GSTA3) mRNA, complete cds 1
Rattus norvegicus growth response protein (CL-6) (LOC64194), mRNA. 0.00124
C1-tetrahydrofolate synthase 0.22
Rat messenger encoding alpha-1-acid glycoprotein 4.47E-05
Mus musculus RIKEN cDNA 3222401G21 gene (3222401G21Rik), mRNA. 0.16
EST225943 Normalized rat brain, Bento Soares Rattus sp. cDNA clone RBRDF91 3' end, mRNA sequence 0.24
Homo sapiens uncharacterized hematopoietic stem 0.02
cDNA encoding chytochrome P-450 from Rat Liver. 2.00E-35
NM_012851 Rattus norvegicus Hydroxysteroid dehydrogenase 17 beta, type 1 (Hsd17b1), mRNA 7.78E-14
Rattus norvegicus rhoB gene (Arhb), mRNA. 6.03E-05
MIF=macrophage migration inhibitory factor [rats, liver, mRNA, 525 nt]. 0.0052
Homo sapiens cDNA FLJ11341 fis, clone PLACE1010786. 0.13
Clinical Gene Expression
(lesion/treatment)
with psoriasis
(lesion/treatment)
Fold Change
Fold Change
• 142 found to associate 1
with improvement of
psoriatic skin in
response to therapeutic
agents
0.1 0.1
• Gene expression 0 1 4 8 12 0 1 4 8 12
• Favorable aspects
– Lowering of risk in conducting high-density
pharmacogenomic studies
– Evaluation by qualified experts
– Consistent evaluation covering multiple
compounds
– Pharmacogenomics review independent of
medical review timeline
– Joint FDA/Industry effort to provide common
basis for research exemption process
Commentary on Preliminary FDA Proposal on
Submission of Pharmacogenomic Data
• Uncertain aspects
– Definition of “Pharmacogenomic Data”
– Terms under which public health concerns
would overrule research exemption
– Process for feedback from FDA to companies
• Unfavorable aspects
– Possible future rescinding of research
exemption
– Potential requirement for additional studies for
drug registration
Key Challenges in Pharmacogenomics
Data submission is only one of many issues
facing industry
• Complexity of biological responses: genetics
isn’t everything
• Value of a pharmacogenomic study is often
unknown until it has been completed
• No clear regulatory pathway for pharmaco-
genomics (including assays)
• Financial constraints weigh against
programs with uncertain outcomes
Unresolved Issues in Application of
Pharmacogenomics