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