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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
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
Best guess
Most diseases are probably interactions between polygenic heritable events, and environmental pressures leading to somatic epigenetic changes.
Translation: diseases are complicated.
Gene by Environment Interaction
DNA
Predisposition Event Disease
FAPMSHBRCALDLr
hydrocarbonsradiationestrogenslow fiber
colon CAcolon CAbreast CAatherosclerosis
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.
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)
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)
Base-pairing (the stuff of life)
T
CA
G
TC
A
G
Lewin. Genes VII page 8.
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.
Melting Curves-Tm measured
TmTm
PCR Primer design
www.oligo.net
Array Choice Factors
Expression profiling:
Sequence known? Not known?
Oligo arrays cDNA arrays
High confidence Clone drift/cross hyb
Immediate ID sequence clones
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.
Laser Capture Microdissection
The importance of purity
Human colon cancer
Blue are normal cells
Red are tumor cells
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
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
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
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´
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
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
Affymetrix® Instrument System Platform for GeneChipPlatform for GeneChip®® Probe Arrays Probe Arrays
• IntegratedIntegrated
• Easy to useEasy to use• ExportableExportable
•VersatileVersatile
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
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
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
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
Simple fold changes• Crude, insensitive--but effective
Criteria:
Present
1.5-fold up/down
Hierachical clustering
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
Pathways of genetic information
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
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
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
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
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
CardioChipin silico workup
Lipoprotein genes/variants
Atherosclerosis markers
Heart failure predictors
Restenosis markers
Coagulation factors
Stress markers
Inflammatory markers
Infectious agents