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Hutchinson-Guilford Progeria
-premature aging-lifespan = 13.4 years-retarded growth-midface hypoplasia-micrognathia-alopecia-low adiposity-osteodysplasia-premature, severe
atherosclerosis-death due to MI
De Sandre-Ciovannoli, Science express, 17 April 2003
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Lamin A mutations in HGS
Exons 11 and 12 code the Lamin A tail (not lamin c)Red is coiled-coil and blue is globular domains1824C>T is aa conservative (G608G) but - in 300 con.1824C>T creates a cryptic donor site at 1819, -50 aa del
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Best guess
Most diseases are probably interactions between polygenic heritable events, and environmental pressures leading to somatic epigenetic changes.
Translation: diseases are complicated.
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Gene by Environment Interaction
DNA
Predisposition Event Disease
FAPMSHBRCALDLr
hydrocarbonsradiationestrogenslow fiber
colon CAcolon CAbreast CAatherosclerosis
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Microarrays-the big net.Ideal disease-hunter: genomic scale protein quantitation andsequencing.
Imperfect solution A: genomic scale detection of mRNA level.Problem: little information on protein level
Imperfect solution B: genome-wide SNP/haplotype.Problem: statistical limits on patient populations
Common compromise: microarray profiling mRNA transcripts(transcript profiling) to identity target areas. Target genesare then followed by proteomics and SNPs.
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Array flavors
DNA detection (SNP, genotyping, etc.)• short oligonucleotides to detect mismatches
RNA detection (transcript profiling)• Plasmid• Inserts• Long oligonucleotides (60 mers)• Short oligonucleotides (20 mers)
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Hybridization-basic elements
• Hybridization = Annealing - Melting
• CRUCIAL: non-covalent, hydrogen bonds
-->equilibrium rules, binding is statistical
• Best hybridization occurs with:• long sequences (no hyb when nt<4)
• high salt concentration (hybrids melt in water)
• low temperatures (hybrids melt with heat)
• G and C (3 H) bind better than A and T (2 H)
• self-complementarity is low (high GC is bad)
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Base-pairing (the stuff of life)
T
CA
G
TC
A
G
Lewin. Genes VII page 8.
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Tm-a good thing.
Tm is a measure of the stability of DS-DNA under a given set of conditions. Stability, and therefore Tm, is affected by:
Strand length - the longer the strand, the higher the TmBase Composition - higher the GC content, the higher the Tm.Ionic Strength - as the ionic strength increases, so does Tm.
Double helical DNA is stabilised by cations. Divalent cations (eg Mg2+) are more effective than
monovalent cations (+ or K+). Organic Solvents - formamide for instance lowers the Tm by weakening the hydrophobic interactions.
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Melting Curves-Tm measured
TmTm
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PCR Primer design
www.oligo.net
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Array Choice Factors
Expression profiling:
Sequence known? Not known?
Oligo arrays cDNA arrays
High confidence Clone drift/cross hyb
Immediate ID sequence clones
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Sample selection-isolate the purest phenotypic examples of test and control
-laser capture microdissection (LCM)-always control for treatment and manipulation-people are the most meaningful, but least controllable-animals are highly controllable, but less meaningful-cell systems (in vitro) are controlled, but meaningful?-small amounts of RNA can be amplified-while purifying cells is good, the processing is bad.
-The quality of the results are directly proportional to the samples that are chosen.
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Laser Capture Microdissection
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The importance of purity
Human colon cancer
Blue are normal cells
Red are tumor cells
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Assessing sample qualityAmount > 5 ug total RNA or 500 ng of poly A+
Basic: O.D. 260/280 ratio >2.1, nucleic acids absorb at 260, protein at 280 nmthus, increasing impurity reduces ratio
Better: agarose gel electrophoresis, EtBR stainedif total RNA, 28s = 2 x 18s ribosomal (Lab-on-
chip)or
Q-PCR of a low and high gene, against standard
Best: test chip
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GeneChip® Probe Arrays
Image of Hybridized Probe ArrayImage of Hybridized Probe Array
1.28cm1.28cm
GeneChipGeneChip Probe ArrayProbe Array
Millions of copies of a specificMillions of copies of a specificoligonucleotide probeoligonucleotide probe
Single stranded, Single stranded, labeled RNA targetlabeled RNA target
Oligonucleotide probeOligonucleotide probe
**
**
*Hybridized Probe CellHybridized Probe Cell
11 µm
>1 million probes
George Washington
Genomics Core Facility
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Synthesis of Ordered Oligonucleotide Arrays
O O O O O
Light(deprotection)
HO HO O O O T T O O O
T T C C O
Light(deprotection)
T T O O O
C A T A TA G C T GT T C C G
MaskMask
SubstrateSubstrate
MaskMask
SubstrateSubstrate
T –T –
C –C –REPEATREPEAT
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GeneChip® Expression Array Design
GeneGeneSequenceSequence
Probes designed to be Probes designed to be Perfect MatchPerfect Match
Probes designed to be Probes designed to be MismatchMismatch
Multiple Multiple oligo probesoligo probes
5´5´ 3´3´
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Procedures for Target Preparation
cDNAcDNAFragmentFragment(heat, Mg(heat, Mg2+2+))
LL LL LL LL
Wash & StainWash & Stain
ScanScan
HybridizeHybridize
(16 hours)(16 hours)
Labeled transcriptLabeled transcript
Poly (A)Poly (A)++
RNARNA
AAAAAAAA
IVTIVT
(Biotin-UTP(Biotin-UTPBiotin-CTP)Biotin-CTP)
Labeled fragmentsLabeled fragments
LL LL
LL
LL
CellsCells
Streptavidin-Phycoerythrin (SAPE)Fluorescent stain-laser stimulated
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A single, contiguous gene set for the rat B-actin gene.
Perfect Match (PM)
Mis Match (MM) Control
PM - MM = difference score
All significant difference scores are averaged to create “average difference” = expression level of the gene.
Each pixel is quantitated and integrated for each oligo feature (range 0-25,000)
Analysis of expression level from probe sets
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Affymetrix® Instrument System Platform for GeneChipPlatform for GeneChip®® Probe Arrays Probe Arrays
• IntegratedIntegrated
• Easy to useEasy to use• ExportableExportable
•VersatileVersatile
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Prepare highly purified RNAO.D. 260/280 = 2.0
Reverse transcribe w/poly dT + T7 = cDNATranscribe with T7 + biotin dUTP = cRNA
Purify probe/hybridize to chip
Wash and detect with avidin/PE + ab amplification
Read fluorescent labelAnd deconvolve genes
Dissect normal media from atherosclerotic lesion
GeneChip analysis of human atherosclerosis
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0.01 0.1 1 10 100 1000 10000
Sample E145 P4-N (raw)0.01
0.1
1
10
100
1000
10000
E145 P22-N (raw)
Basic Bioinformatics-Scatterplot
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Transcript profiling of aged rat aorta.Affymetrix GeneChip analysis of 10 aortas @ 20 mo. vs. 3 mo.
mRNAs Decreased in the Aged AortaExperiment 1 Experiment 2 DescriptionsSignal Change Signal Change
12884 -3.6 14901 -4.9 Egr-1 (3 probe sets)9440 -5.7 25730 -3.5 collagen alpha1 type I (3 probe sets)
342 -3.3 330 -3.9 flavin-containing monooxygenase 1 (FMO-1)7540 -2.8 5989 -3.3 cyclooxygenase isoform COX-21781 -3.4 2053 -3.5 leucine zipper protein mRNA3090 -8.2 2291 -3.1 heat shock protein 70 (3 probe sets)1718 -3.1 3836 -3.0 DNA polymerase alpha 6897 -2.3 4514 -2.7 phosphoenolpyruvate carboxykinase (GTP)3552 -2.9 3284 -2.2 retinol-binding protein (RBP) 9063 -2.1 6580 -2.0 C4 complement protein 1372 -6.5 6282 -1.9 DnaJ-like protein (RDJ1) 8604 -2.2 10106 -2.2 plasminogen activator inhibitor-1 (PAI-1) 3845 -4.5 13019 -1.7 RCO4-1 gene for cytochrome c oxidase subunit IV1708 -11.1 11044 -1.6 lipoprotein lipase 6593 -2.1 12816 -1.5 RTK40 homolog3139 -2.1 7395 -1.5 ribonucleoprotein F
* * * * AND 18 ESTs
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FAQs: How many replicates?Number of Genes Called Differentially Expressed as a Function of
Number of Replicates
0
500
1000
1500
2000
2500
3000
3500
4000
4500
1 2 3 4 5 6 7
Number of Replicates
Number of Genes Greater Than 2 Fold
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Simple fold changes• Crude, insensitive--but effective
Criteria:
Present
1.5-fold up/down
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Hierachical clustering
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Statistical testing and ontologyGene Abbrev. Fold Lists Description Gene Abbrev. Fold Lists Description
Apoptosis Growth factors/regulatorsBAD 1.4 * BCL2-antagonist of cell death FGF5 2.3 ** fibroblast growth factor 5BCL2L1 6.6 * BCL2-like 1 (BCL-XL) HDGF -1.2 *** hepatoma-derived growth factor (high-mobility group protein 1-like)CCND1 1.9 *** cyclin D1, PRAD1 IGFBP3 -1.6 ** insulin-like growth factor binding protein 3 (2 sets)MDM2 2.2 * Mdm2, p53 binding protein IGFBP4 -1.5 * insulin-like growth factor binding protein 4PRSS25 ? ? serine protease 25-Omi/HtrA2 LRP1 -1.7 ** LRP1, TGF-ß Type V receptorTNFRSF6 1.2 *** TNF receptor superfamily, 6, fas, CD95 LTBP2 -1.3 *** latent transforming growth factor beta binding protein 2VDAC2 1.8 *** voltage-dependent anion channel 2 SMURF2 1.8 *** SMAD-specific ubiquitin ligase
VEGFB -1.5 *** vascular endothelial growth factor BCell CycleCCND1 1.9 *** cyclin D1, PRAD1 (3 sets) SignallingCCNI -1.6 *** cyclin I FKBP9 -2.4 * FK506 binding protein 9, 63 kDaCDK11 -1.6 *** cyclin-dependent kinase (CDC2-like) 11 JAK1 -1.2 * Janus associated kinase 1CUL1 -1.3 ** cullin 1-cyclin D1 degrading MAP3K12 -1.7 *** mitogen-activated protein kinase kinase kinase 12JUN 1.4 *** v-Jun homolog MAP3K4 -1.4 ** mitogen-activated protein kinase kinase kinase 4MDM2 2.2 * Mdm2, p53 binding protein PPIH 3.4 * peptidyl prolyl isomerase H (cyclophilin H)PDGFRB -2.1 ** platelet-derived growth factor receptor, beta STAT1 1.4 ** signal transducer and transactivator 1
STAT3 -1.4 * signal transducer and transactivator 3Chromatin remodeling STAT6 -1.3 *** signal transducer and transactivator 6CBFA2T1 -1.6 * core-binding factor, cyclin D-relatedCHD3 -1.5 *** chromodomain helicase DNA binding protein 3 Mitochondrial/MetabolicHDAC4 -1.5 * histone deacetylase 4 AHCYL1 -1.5 *** S-adenosylhomocysteine hydrolase-like 1 (3 sets)HIST1H2BN 1.8 ** histone 1, both H2bn and H2bd ATP5J 1.2 *** ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F6HIST1H2AL 1.7 ** histone 1, H2al ETFA 1.3 *** electron-transfer-flavoprotein, alpha polypeptide (glutaric aciduria II)MYST1 -1.5 *** MYST histone acetyltransferase 1 HCCS 1.4 *** holocytochrome c synthase (cytochrome c heme-lyase)POLB 1.8 *** polymerase (DNA directed), beta TOMM34 1.4 *** translocase of outer mitochondrial membrane 34
Cholesterol/Fatty acid/Membranes Stress/oxidant/antioxidantATP8B1 2.3 *** Potential phospholipid-transporting ATPase DNAJA2 1.3 ** DnaJ (Hsp40) homolog, subfamily A, member 2FADS1 -1.4 ** fatty acid desaturase 1 DNAJB4 2.7 *** DnaJ (Hsp40) homolog B4 (2 sets), HLJ1LRP1 -1.7 ** low density lipoprotein-related protein 1 PSMF1 1.4 * proteasome (prosome, macropain) inhibitor subunit 1 (PI31)PLTP -1.4 *** phospholipid transfer protein PSMB6 1.4 * proteasome (prosome, macropain) subunit, beta type, 6SRD5A1 1.9 ** steroid-5-alpha-reductase, alpha 1 PTMA -1.5 ** prothymosin, alpha (gene sequence 28)
SOD3 -1.6 ** superoxide dismutase 3, extracellularExtracellular MatrixCOL1A2 -1.3 *** collagen, type I, alpha 2 (2 sets) Transcription factorsCOL6A1 -1.6 *** collagen, type VI, alpha 1 BLZF1 1.8 *** basic leucine zipper nuclear factor 1 (JEM-1)FBN1 -1.3 *** fibrillin 1 (Marfan syndrome) CEBPD -1.4 ** CCAAT/enhancer binding protein (C/EBP), deltaFN1 -1.3 ** fibronectin 1 (2 sets) JUN 1.4 *** v-Jun homologLAMB2 -1.4 *** laminin, beta 2 (laminin S) MSC -1.5 *** musculin (lamin C homolog, repressor)LAMA2 -1.6 *** laminin, alpha 2 (merosin) ZNF24 -1.3 *** zinc finger protein 24 (KOX 17)RECK ? *** reversion-inducing cys-rich w/Kazal (MMP9 regulator) ZNF42 1.4 * zinc finger protein 42 (myeloid-specific retinoic acid- responsive)TIMP1 -1.5 ** tissue inhibitor of metalloproteinase 1 (2 sets) ZNF337 -1.3 * zinc finger protein 337
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Pathways of genetic information
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Expression of Egr-1 mRNA in human lesions.
L M5 65
E213 E217
H20
Egr-1
RhoA
L M
Patient #
MinutesTissue L M
5 65L M
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B)
M L M LE240
M LE221E197
Egr-1
Actin
Western blot
M LE243
0
5
10
15
20
E197 E196
LesionMedia
A)
xx
Egr-1 mRNA and protein in lesions vs normal cells.
Egr
-1 m
RN
A
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Expression screening by GeneChip
• each oligo sequence (20 mer) is synthesized as a 11 µ square (feature)
• each feature contains > 1 million copies of the oligo• scanner resolution is about 2 µ (pixel)• each gene is quantitated by 11 oligos and
compared to equal # of mismatched controls• 44,000 genes are evaluated with 11 matching oligos
and 11 mismatched oligos = 4 x 106 features/chip• features are photolithographically synthesized
onto a 2 x 2 cm glass substrate
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GeneChip® Array Advantages – Specificity
Gene “on”Gene “on”
Gene “off”Gene “off”
Oligo arraysOligo arrays cDNA arrayscDNA arrays
Detection PatternDetection Pattern Single SpotSingle Spot
24 µm24 µm
~ 150 µm~ 150 µm
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Limitations to all microarrays.
- dynamic range of gene expression:very difficult to simultaneously detect low and high abundance genes accurately
- each gene has multiple splice variants 2 splice variants may have opposite effects (i.e. trk)arrays can be designed for splicing, but complexity ^ 5X
- translational efficiency is a regulated process:mRNA level does not correlate with protein level
- proteins are modified post-translationallyglycosylation, phosphorylation, etc.
- pathogens might have little ‘genomic’ effect
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CardioChipin silico workup
Lipoprotein genes/variants
Atherosclerosis markers
Heart failure predictors
Restenosis markers
Coagulation factors
Stress markers
Inflammatory markers
Infectious agents