Optimization of differential expression analysis in genetic
disease : Cystic Fibrosis.
Voisin GrégoryLemieux’s lab -IRIC
February 2007.Codirected by Dr Yves Berthiaume
(CRCHUM)
… 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.
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