Optimization of differential expression analysis in genetic

disease : Cystic Fibrosis.

Voisin GrégoryLemieux’s lab -IRIC

February 2007.Codirected by Dr Yves Berthiaume

(CRCHUM)

A few words about…

… Cystic Fibrosis (CF)

Clinical symptoms :

• Accumulation of mucus in lungs.• Obstruction of pancreatic and hepatic ducts.• Fibrosis of tissues. • The hallmark:

chronic infection (P. aeruginosa) and EXCESSIVE INFLAMMATION.

… CFTR

• CFTR: Cystic Fibrosis Transmembrane conductance Regulator.

• Member 7 of ATP binding cassette transporter family.

• Critical Physiological Function: Chlorure ionic Channel.

• + 1000 mutations (most frequent = ΔF508 ) .

… GeneChips AFFYMETRIX

• Based on hybrizidation mRNA/probe

• Human Genome U133.2 +:

47 000 transcripts and variants, (38 500 well characterized human genes).

=

54675 numeric Data for exploitation

Problematics

• Bioinformatics problematic: establish a robust methodology to allow effective data-mining.

• Molecular and biological problematic: understand the molecular regulation of inflammation.

• Clinical problematic: understand the developpement, inflammation installation of disease.

Our microarray experiment

Hypothesis

(Inflammatory) genes regulation <=> CFTR ?

CFTR deficiency in Human Alveolar Epithelium cells triggers specific pathways

involved in the inflammatory response.

.CEL

RNA extraction

Nuli cells:Normal Lung

Cufi cells:

Cystic Fibrosis, (homozygote ∆F508)

MethodologyHybridation

Scanning by bioanalyzer

Data acquisition

Normalizationby RMA express

Statistical analysiswith Bioconductor (AffyLM package)

DEGs

Observation

Pathway-express

Onto-expressInteresting

DEGs

Biological questionQ-PCR

Protein expression

Pathway activation

Promoter analysisNew elements about

biological question DEGs: Differential Expressed Gene

• Differential expression analysis.

• Biological processes modulated.

• Metabolic pathways modulated.

• Promotor-oriented Analysis.

Specific Objectives

ResultsDEGs

2335 PROBESETS differentially expressed.

Observation1659 annotated DEGS.

871 DEGs DOWN-regulated

788 DEGs UP-regulated

202 genes NA +

474 n-plicate

Pathway-expressToll-like receptor Signaling PathwayJak-Stat signaling Pathway

Onto-express

Immune response, inflammatory response, chemotaxis, signal transduction, transport, lipid metabolism.

Q-PCR28 tested genes.Confirmed expression: IL1b, IL8, IL6, CXCL10 ,CXCL11,CLCA4, KCNK5, STAT1, GSTT1.

Protein expression

10 tested proteins. Confirmed expression: CXCL11, IL8, 10,15, CCL 2,8, 7.

Interesting DEGs

Microarray modulated genes in Toll-like Receptor Signaling Pathway.

0

1

2

3

4

5

6

7

Nuli Cufi Nuli Cufi Nuli Cufi Nuli Cufi Nuli Cufi

IL8 IL1B IL6 CXCL10 (IP-10) CXCL11(ITAC)

MYD88Pvalue= 0,88 , ratio =1,1

0

0,2

0,4

0,6

0,8

1

Nul i CufiSTAT1Pvalue = 0.006 , expression ratio= 2.1

0,0

0,5

1,0

1,5

2,0

2,5

3,0

Nuli Cufi

Microarray modulated genes in Jak-Stat Signaling Pathway.

SOCS

0,0

2,0

4,0

6,0

8,0

10,0

12,0

14,0

Nuli Cufi Nuli Cufi Nuli Cufi Nuli Cufi Nuli Cufi Nuli Cufi

SOCS1 SOCS2 SOCS3 SOCS4 SOC5.VAR1 SOC5.VAR2

Pv= 0,0004

Pv= 0,067 Pv= 0,13

Pv= 0,15 Pv=0,19 Pv= 0,78

STAT and PIAS

0,0

0,5

1,0

1,5

2,0

2,5

3,0

Nuli Cufi Nuli Cufi Nuli Cufi Nuli Cufi Nuli Cufi Nuli Cufi

STAT1 STAT2 PIAS1 PIAS2 PIAS3 PIAS4

Pv= 0,0006

Pv= 0,03

Pv= 0,8Pv= 0,03 Pv= 0,25

Pv= 0,05

Non-inflammatory gene validated by QPCR

00,5

11,5

22,5

33,5

44,5

5

Nuli Cufi Nuli Cufi Nuli Cufi

GSTT1 KCNK5 CLCA4

oxidatif stress ionic channel

Pv= 0,0001Pv= 0,003 Pv= 0,003

Glutathione S-transferase (GST) theta 1 (GSTT1) is a memberof a superfamily of proteins that catalyze the conjugation of reduced glutathione to a variety of electrophilic and hydrophobic compounds.

KCNK5This gene encodes one of the members of the superfamily of potassium channel proteins .

The protein is highly sensitive to external pH.

CLCA4The protein encoded by this gene belongs to the calcium sensitive chloride conductance protein family.The exact function of this protein is not known.

Interpretations

• In the absence of pathogen agents, we observe an up-regulation of several inflammatory actors in Cufi cells.

• CFTR deficiency could be responsible for an excessive immune and inflammatory response.

• There could be a dysregulation of TLR and Jak-Stat Signaling pathway in CF cells.

Work in progress

Pathway activation

Promoter analysis

•Selection of 96 modulated genes with GO = inflammatory response, immune response,chemotaxis.

•Analysis of 2200 bp of these promoters to find Transcription Factor (TF) sites or module of TF sites over represented.

Find a specific inhibitor and validate pathway activation.

Limitations of our optimization

• Over 1500 modulated genes.

• Inconsistent and incomplete annotations.

• Concepts used in methodology produce a technical bias.

• Transcriptomics analysis only.

RNA extraction

Nuli cells:Normal Lung

Cufi cells:

Cystic Fibrosis, (homozygote ∆F508)

Future workhybridation

Scanning by bioanalyzer

Data acquisition

Normalizationby RMA express

Statistical analysiswith Bioconductor (AffyLM package)

.CEL

DEGs

Cut

off

Observation !!

Pathway-express

Onto-express

Interest DEGs

Biological questio

n

Q-PCR

protein expression

Pathway activation

Promoter analysis

Conclusion about

biological question

Others microarray experiments

Same problematic.Same technology

Different modelDifferent organismDifferent design

+ experimental conditions : oxydant

Berthiaume

Xu

RadziochVirella-Lowell

Zabner Wright

.CEL.CEL.CEL.CEL.CEL.CEL.CEL.CEL.CEL.CEL

Normalizationby RMA express Statistical analysiswith Bioconductor (AffyLM package)

DEGsDEGsDEGsDEGsDEGsDEGs

Comparaison

Conclusion more precise

about

biological question

1. Zabner J, Scheetz TE, Almabrazi HG, Casavant TL, Huang J, Keshavjee S, McCray PB Jr.CFTR DeltaF508 mutation has minimal effect on the gene expression Am J Physiol Lung Cell Mol Physiol. 2005 Oct;289(4):L545-53. Epub 2005 Jun 3. 2. Wright JM, Merlo CA, Reynolds JB, Zeitlin PL, Garcia JG, Guggino WB, Boyle MP. Respiratory epithelial gene expression in patients with mild and severe cystic fibrosis lung disease.Am J Respir Cell Mol Biol. 2006 Sep;35(3):327-36. Epub 2006 Apr 13. ] 3. Xu Y, Liu C, Clark JC, Whitsett JA.Functional genomic responses to cystic fibrosis transmembrane conductance regulator (CFTR) and CFTR(delta508) in the lung.J Biol Chem. 2006 Apr 21;281(16):11279-91. Epub 2006 Feb 2. 4. Guilbault C, Novak JP, Martin P, Boghdady ML, Saeed Z, Guiot MC, Hudson TJ, Radzioch D. Distinct pattern of lung gene expression in the Cftr-KO mice developing spontaneous lung disease compared with their littermate controls.Physiol Genomics. 2006 Apr 13;25(2):179-93. Epub 2006 Jan 17. 5. Virella-Lowell I, Herlihy JD, Liu B, Lopez C, Cruz P, Muller C, Baker HV, Flotte TR. Effects of CFTR, interleukin-10, and Pseudomonas aeruginosa on gene expression profiles in a CF bronchial

epithelial cell Line. Mol Ther. 2004 Sep;10(3):562-73.

Acknowledgements• Dr Sébastien Lemieux.

• Dr Yves Berthiaume.

• Dr André Dagenais.

• Members of Berthiaume’s Lab.

• Members of Lemieux’s Lab.

• IRIC’S Genomics Platform.

EXTRA : Range values

0.01<Adjusted Pvalues<10℮-130.5<Expression probability<10.006<Expression Ratio <19

0

1

2

3

4

5

6

7

Nuli Cufi Nuli Cufi Nuli Cufi Nuli Cufi Nuli Cufi

IL8 IL1B IL6 CXCL10 (IP-10) CXCL11(ITAC)

RATIO qPCR 3,6

EXPRES.PROBAB.MICROARRAY

0,002

RATIO MICROARRAY

P-value qPCR

4

>95 %

4,7

0,0004

2

>95 %

5,4

0,01

17

>95 %

5,5

0,028

9

>95 %

4,9

0,02

18

>50%

EXTRA: :Confirmation by QPCR of gene expression .CUFINULI

Analyse ontologique….

...sur ...sur l’ensemblel’ensemble des probesets des probesets

modulésmodulés

...sur l’ensemble des...sur l’ensemble des1296 probesets down-regules1296 probesets down-regules

...sur l’ensemble...sur l’ensemble 1039 probesets1039 probesets up-regulesup-regules

MECANISME DE DEFENSE:immune responseinflammatory response

COMMUNICATION CELLULAIRE:cell-cell signalingchemotaxiscell adhesionMETABOLISME:lipid metabolism

SIGNAL DE TRANSDUCTION:Cell surface receptor linked signal transduction Positive regulation of I-kappa bIntracellular signaling cascade.

METABOLISME:lipid metabolismprotein biosynthesis

MODIFICATION DE PROTEINE:protein amino acid phosphorylationProteine byosyntheseTRANSCRIPTION:

TRANSPORTelectron transport

MECANISME DE DEFENSE:immune responseinflammatory response

COMMUNICATION CELLULAIRE:cell-cell signalingchemotaxis.cell adhesion

METABOLISME:lipid metabolismprotein biosynthesis

VOIE DE SIGNALISATION

TRANSPORTelectron transport

Activation of NF-kappa B in Cufi and Nuli Cells

nuclear P50 NF-kappaB

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

Ctrl-P15 Ctrl-P16

DO

450

nm

/650

nm

Nuli

Cufi

p65 NF-kB Cufi Nuli

Ctrl LPS TNF0

150

300

450

600

750

900NuliCufi

Conditions

%/C

trl

Nu

li

Hypothesis :

Another system of TF could

modulated inflammatory genes

Empirical Bayes log posterior odds statistic

•The B statistic is the log odds of differential expression (also known as a lod score). Bascially if it is greater than 0 then you have more than a 50/50 chance that your gene is truly differentially expressed given its fitted M value