combine gx and qtl analysis - university of leicester · 19 29.3 lipk lipase, family member k 19...
TRANSCRIPT
02/10/2012
1
Magdalena Jonczyk, Vitor Fernandes, Nicolas Sylvius, Paul Denny, Peter Andrew
PNEUMOPATH, Leicester University, UK
Summary
Problem
Genetic control of susceptibility to pneumococcal infection
Methods
Phenotyping
QTL mapping
Gene identification
Results
Susceptibility QTLs in mice model
Candidate genes
Background
Different strains of inbred mice show different susceptibility to pneumococcal diseases
Some of proteins and cells involved in immune responses and inflammation could modulate clinical outcome of the disease
Polymorphism in several human genes associated with increase susceptibility to pneumococcal diseases
QTL mapping
Congenic mice lines
Very costly
Requires long-term breeding program
Limited number of QTL can be studied
Large QTL intervals
In silico mapping (GWAS)
Availability of single nucleotide polymorphism (SNP) data for inbred mouse strain (no genotyping required)
Relatively cheap and quick
Usually small QTL intervals
Spir1: QTL controlling susceptibility to pneumonia (Denny et al 2003)
Spir1 region was identified on chromosome 7 by congenic mouse strains (Denny et al 2003)
QTL interval of about 7cM (between 25Mb and 40Mb)
Methods
Laboratory Phenotyping of genetically defined inbred mouse strains
(Jackson Laboratories, USA)
Transcriptional profiling of resistant and susceptible mouse strains at early stage of disease (6h, Illumina platform)
Bioinformatics Genome Wide Association Analysis (GWAS) of mouse
phenotype data using two statistical models:
Efficient Mixed Model Association (EMMA)
Haplotype Association Model (HAM)
Selection of genes within QTLs (MPD)
Selection of differentially expressed genes (ArrayTrack)
Meta-analysis of gene expression and QTL data – selection of gene candidates
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Results
Phenotypes of 26 Jax-mouse strains
Phenotypes of 26 Jax-mouse strains Phenotyping data SNP data
GWAS analysis EMMA
PLINK (HAM)
Gene expression
QTL
Combined : P(value) , phenotype, model Stringent P(value)
Selection of QTL
Gene list
QTL
G e n e c a n d i d a t e s
Gene list Gene list
QTLs report
No QTLs Min P No QTLs Min P No QTLs Min P No QTLs Min P No QTLs Min P
All
chromosomes12 5.34E-06 12 1.09E-05 12 1.52E-07 13 1.82E-06 21 5.56E-06
Clinical Lung Survival ToD 24h
HAM
No QTLs Min P No QTLs Min P No QTLs Min P No QTLs Min P No QTLs Min P
All
chromosomes23 2.62E-09 30 7.92E-09 22 1.68E-10 37 6.14E-09 3 1.69E-06
24hClinical Lung Survival ToD
EMMA
No QTLs Min P No QTLs Min P No QTLs Min P No QTLs Min P No QTLs Min P
1 1 3.83E-06 1
2 2 2.88E-06 2 7.09E-06 1 7.44E-07 1 6.27E-06 2
4 1 2.62E-09 3 1.65E-08 1 2.54E-10 3 1.59E-08 4
5 3 9.08E-06 2 1.11E-06 1 6.70E-08 1 4.69E-07 5
6 1 3.63E-06 1 9.07E-07 6
7 7 2.70E-07 7 1.63E-07 5 2.51E-08 9 2.57E-07 1 1.69E-06 7
9 2 1.43E-06 9
10 2 9.21E-07 2 1.26E-05 10
12 2 1.35E-05 12
13 1 5.19E-06 7 6.47E-07 3 4.74E-07 6 2.57E-07 13
15 2 4.12E-07 1 7.62E-06 1 3.97E-06 2 4.56E-07 15
16 2 2.06E-06 2 4.76E-07 2 7.98E-08 2 1.95E-07 16
18 1 1.76E-07 1 1.16E-08 1 5.63E-06 18
19 3 2.85E-07 4 7.92E-09 5 1.68E-10 6 6.14E-09 2 3.85E-06 19
chrom
EMMA
Clinical Lung Survival ToD 24hchrom
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No QTLs Min P No QTLs Min P No QTLs Min P No QTLs Min P No QTLs Min P
1 1 4.17E-05 1 1.57E-05 3 5.56E-06 1
2 1 5.34E-06 1 3.54E-05 1 4.05E-05 1 3.05E-05 2
3 1 4.85E-05 1 6.14E-06 3
5 1 4.46E-05 5
6 1 8.93E-06 1 6.29E-06 2 1.78E-05 6
7 6 1.71E-05 2 2.75E-05 2 7.37E-06 2 1.30E-05 3 1.62E-05 7
9 2 2.70E-05 1 3.92E-05 9
10 1 3.78E-05 1 1.79E-05 1 2.22E-05 10
11 2 8.11E-06 11
12 2 3.39E-05 12
13 3 1.09E-05 2 5.97E-06 1 2.69E-05 2 1.66E-05 13
16 1 4.89E-05 1 1.32E-05 1 1.38E-05 1 4.52E-05 16
17 1 3.85E-05 2 1.68E-05 17
18 1 3.42E-05 1 4.96E-07 18
19 1 3.65E-05 3 1.06E-05 4 1.52E-07 2 1.82E-06 2 6.05E-06 19
chrom
PLINK (HAM)
Lung Survival ToD 24hchrom
Clinical
Combined approach
Criteria for a QTL to be selected: Significant in at least 4 tested phenotypes
Significant in both statistical models
Hypothesis: Susceptibility to infectious diseases is controlled by multiple
QTLs
If dominant QTL exist it might be selected by all or majority of phenotypes
If the QTL is confirmed by more than one statistical model it is more likely to be true QTL
QTL result comparison for EMMA and HAM (PLINK)
Pain score
PLINK
19
EMMA
1 2
PLINK
7
EMMA
31 6
PLINK
5
EMMA
15 7
PLINK
4
EMMA
22 8
24h bacteremia
ToD bacteremia
Survival
Bacteria in Lungs
EMMA and HAM comparison for each phenotype
position
(Mb)
No of
SNPs
position
(Mb)
No of
SNPs
position
(Mb)
No of
SNPs
position
(Mb)
No of
SNPs
position
(Mb)
No of
SNPs
1 162.6 3
2 132 6
6 44-45 32
7 30 1 30 1
9 116.7 1
12 143.1 3
13 59.1 2 59.2 2
13 67.6 3
16 58.4 1 58.4 1 58.4 1
18 73.4 4 73.4 4
19 20 2
19 25.6 1 25.6 1
19 35 2 34-35 16 34-35 14 34-35 7 35 2
19 41.3 1 41.3 1 41.3 1
19 46.3 1
Total 13 26 24 47 4
Chrom
Clinical Lung Survival ToD 24h
Genes within selected QTL
Chr cM GENENAME Gene Expression Description
19 29.76 2010002M12Rik RIKEN cDNA 2010002M12 gene
19 29.41 Acta2 actin, alpha 2, smooth muscle, aorta
19 29.33 Ankrd22 ankyrin repeat domain 22
19 29.69 Ch25h upregulated cholesterol 25-hydroxylase
19 29.48 Fas upregulated Fas (TNF receptor superfamily member 6)
19 29.76 I830012O16Rik RIKEN cDNA I830012O16 gene
19 29.78 Ifit1 interferon-induced protein with tetratricopeptide repeats 1
19 29.73 Ifit2 interferon-induced protein with tetratricopeptide repeats 2
19 29.74 Ifit3 downregulated interferon-induced protein with tetratricopeptide repeats 3
19 29.79 Kif20b kinesin family member 20B
19 29.7 Lipa lysosomal acid lipase A
19 29.29 Lipf lipase, gastric
19 29.3 Lipk lipase, family member K
19 29.32 Lipm lipase, family member M
19 29.32 Lipn lipase, family member N
19 29.18 Lipo1 lipase, member O1
19 29.23 Lipo2 lipase, member O2
19 29.17 Lipo4 lipase, member O4
19 29.79 Mir107 microRNA 107
19 29.79 Mir1950 microRNA 1950
19 29.79 Pank1 downregulated pantothenate kinase 1
19 29.79 Slc16a12solute carrier family 16 (monocarboxylic acid transporters),
member 12
19 29.35 Stambpl1 STAM binding protein like 1
Differentially expressed genes within selected QTL
resistant sensitive
19 29.69 Ch25h 34,548,276 34,549,625 7.22 5.69 2.17 cholesterol 25-hydroxylase
19 29.48 Fas 34,365,149 34,402,260 2.75 2.31 1.47 Fas (TNF receptor superfamily member 6)
19 29.74 Ifit3 34,658,021 34,663,221 0.73 0.67 1.50 interferon-induced protein with tetratricopeptide repeats 3
19 29.79 Pank1 34,885,384 34,953,945 0.55 0.64 pantothenate kinase 1
control
S/RDescription
Gene expression
fold changeChr cM GENENAME BP1 BP2
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Consequences of SNP polymorphism within candidate genes
con
seq
uen
ce
Gen
e n
ame
ob
serv
ed
BA
LB/c
ByJ
BA
LB/c
J
A/J
FVB
/NJ
LP/J
NO
D/S
hiL
tJ
C5
7B
L/6
J
12
9S1
/SvI
mJ
AK
R/J
C3
H/H
eJ
CB
A/J
DB
A/2
J
NZW
/Lac
J
I, Reg Acta2 A/G G G G G A A A A A A A A A
Cs Acta2 A/G G G G G A A A A A A A A A
Cn RH Fas G/A A A A A G G G G G G G G G
U3,I Fas C/T T T T T C C C C C C C C
U3,I Fas G/T T T G G G G G G G G
U3,I Fas C/T T T C C C C C C C C
Cs Fas C/T T T C C C C C C C C
Cs Fas G/A A A A A G G G G G G G G G
Cs Fas G/A A A A A G G G G G G G G G
Resistant Susceptible
Result
Only 1 QTL is consistently selected by both models and each tested phenotypes
Fas, Ch25h, Pank1, Acta2 and Ifit3 are the strongest gene candidates
Stringent P value
Criteria for a QTL to be selected: P value of the SNP should be within 10% of top
significant P values (P<5x10-5)
Significant in any statistical model
Significant for any phenotype
Hypothesis: Multiply phenotypes might help mitigate influence of
the biological variance
The highest P values are more likely to indicate true positive QTL
Stringent P-value: QTL selected for both models
chromPosition
(Mb)P-value
4 145-147 2.54E-10
5 148.3 1.23E-07
5 150.4 6.7E-08
7 29-30 2.51E-08
13 59 2.51E-08
16 58 7.98E-08
18 73 1.16E-08
19 25.6 2.51E-08
19 34-35 1.68E-10
19 41.3 2.51E-08
EMMA
chromposition
(Mb)P-value
1 45.8 5.56E-06
2 132.2 5.34E-06
3 38.5 6.14E-06
6 45 6.29E-06
7 30 7.37E-06
13 59 5.97E-06
18 73 4.96E-07
19 20.2 6.05E-06
19 33-35 1.52E-07
HAM
Genetic location of SNPs with the lowest p-Value in any model for any phenotype
Chromposition
MbP-value
4 145-147 2.54E-10
5 148.3 1.23E-07
5 150.4 6.7E-08
7 20.4 2.7E-07
7 29-30 2.51E-08
13 59 2.51E-08
16 58 7.98E-08
18 73 1.16E-08
19 25.6 2.51E-08
19 34-35 1.68E-10
19 41.3 2.51E-08
Spir1: QTL controlling susceptibility to pneumonia (Denny et al 2003)
Spir1 region was identified on chromosome 7 by congenic mouse strains (Denny et al 2003)
QTL interval of about 7cM (between 25Mb and 40Mb)
In silico analysis in this study identified susceptibility loci on chromosome 7 between 29Mb and 30Mb and the second one at 20Mb
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Chromposition
MbP-value
19 25.6 2.51E-08
19 34-35 1.68E-10
19 41.3 2.51E-08
Linkage Disequilibrium within QTL on chromosome 19
chrom 19
25
,57
3,6
88
34
,32
1,8
66
34
,38
0,4
52
34
,46
9,9
06
35
,02
4,2
79
41
,28
6,6
07
41
,41
2,8
14
25,573,688 1 1 1 0.49 0.49 1 1
34,321,866 1 1 0.49 0.49 1 1
34,380,452 1 0.49 0.49 1 1
34,469,906 1 1 0.49 0.43
35,024,279 1 0.49 0.43
41,286,607 1 1
41,412,814 1
Linkage Disequilibrium within QTL on chromosome 7
Chromposition
MbP-value
7 20.4 2.7E-07
7 29-30 2.51E-08chrom 7
19
,98
8,3
55
25
,21
8,9
67
29
,69
6,4
12
30
,00
3,0
76
30
,04
3,6
03
30
,10
1,4
67
19,988,355 1 0.20 0.20 0.23 0.29 0.20
25,218,967 1 1 0.64 0.72 1
29,696,412 1 0.64 0.72 1
30,003,076 1 1 0.64
30,043,603 1 0.72
30,101,467 1
resistant sensitive
19 29.69 Ch25h 34,548,276 34,549,625 7.22 5.69 2.17 cholesterol 25-hydroxylase
19 29.48 Fas 34,365,149 34,402,260 2.75 2.31 1.47 Fas (TNF receptor superfamily member 6)
19 29.74 Ifit3 34,658,021 34,663,221 0.73 0.67 1.50interferon-induced protein with
tetratricopeptide repeats 3
19 29.79 Pank1 34,885,384 34,953,945 0.55 0.64 pantothenate kinase 1
4 78.67 Agtrap 147,451,170 147,462,140 0.26 angiotensin II, type I receptor-associated protein
4 78.17 Tnfrsf1b 144,803,366 144,836,773 1.32 1.97tumor necrosis factor receptor superfamily,
member 1b
5 86.56 Rasl11a 147,656,647 147,659,302 1.63 RAS-like, family 11, member A
5 89.18 6330406I15Rik 150,171,051 150,234,298 2.03 1.86 RIKEN cDNA 6330406I15 gene
5 89.18 Alox5ap 150,076,634 150,076,758 1.82 1.57 arachidonate 5-lipoxygenase activating protein
7 16.67 Dyrk1b 28,964,488 28,972,313 0.78 0.62dual-specificity tyrosine-(Y)-phosphorylation
regulated kinase 1b
7 16.76 Gmfg 29,222,466 29,233,252 1.26 1.66 glia maturation factor, gamma
7 16.91 Mrps12 29,524,660 29,526,839 2.76 mitochondrial ribosomal protein S12
7 16.91 Nfkbib 29,543,270 29,552,531 1.94 1.45nuclear factor of kappa light polypeptide gene
enhancer in B cells inhibitor, beta
7 16.94 Ppp1r14a 30,074,339 30,078,409 0.70protein phosphatase 1, regulatory (inhibitor)
subunit 14A
7 16.94 Psmd8 29,959,207 29,965,692 2.89proteasome (prosome, macropain) 26S subunit,
non-ATPase, 8
7 16.72 Zfp36 29,161,803 29,164,274 2.36 2.64 zinc finger protein 36
7 15.97 Zfp60 28,516,408 28,538,721 0.69 zinc finger protein 60
7 9.94 Apoe 20,281,458 20,284,515 1.72 apolipoprotein E
7 9.95 Bcl3 20,393,811 20,408,119 3.47 5.85 B cell leukemia/lymphoma 3
7 9.95 Pvr 20,488,927 20,506,509 0.41 2.03 poliovirus receptor
7 9.93 Relb 20,191,566 20,214,787 3.47 5.85avian reticuloendotheliosis viral (v-rel) oncogene
related B
BP2
Gene expression
fold change control
S/RDescriptionChr cM GENENAME BP1
Selected top gene candidates
resistant sensitive
19 29.69 Ch25h 34,548,276 34,549,625 7.22 5.69 2.17 cholesterol 25-hydroxylase
19 29.48 Fas 34,365,149 34,402,260 2.75 2.31 1.47 Fas (TNF receptor superfamily member 6)
19 29.79 Pank1 34,885,384 34,953,945 0.55 0.64pantothenate kinase 1
7 16.72 Zfp36 29,161,803 29,164,274 2.36 2.64 zinc finger protein 36
7 15.97 Zfp60 28,516,408 28,538,721 0.69 zinc finger protein 60
7 9.94 Apoe 20,281,458 20,284,515 1.72 apolipoprotein E
7 9.95 Bcl3 20,393,811 20,408,119 3.47 5.85 B cell leukemia/lymphoma 3
7 9.95 Pvr 20,488,927 20,506,509 0.41 2.03 poliovirus receptor
7 9.93 Relb 20,191,566 20,214,787 2.32 3.38avian reticuloendotheliosis viral (v-rel)
oncogene related B
cMChr GENENAME BP1 BP2
Gene expression
fold changecontrol
S/RDescription
Regulatory elements
19 29.79 Mir107 34,895,177 34,895,263 microRNA 107
19 29.79 Mir1950 35,039,886 35,039,959 microRNA 1950
19 19.87 Mir3084 25,016,725 25,016,793 microRNA 3084
7 9.62 Mir343 19,971,992 19,972,066 microRNA 343
7 16.94 Mir1963 29,868,655 29,868,713 microRNA 1963
19 QTL Tsiq2 33,009,697 45,226,509 T cell secretion of IL4 QTL 2
Chr cM GENENAME BP1 BP2 Description
Results
Stringent p-Value criteria allowed selection of 11 QTLs associating with pneumonia susceptibility
Pvr, Ch25h, Fas, Mir107, Bcl3 and Relb are the strongest candidates
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Conclusion
QTL on chromosome 19 shows the strongest association with susceptibility to pneumonia in mice
QTLs on chromosome 7 are located within published Spir1 region
Acknowledgement
PNEUMOPATH Consortium
Dr Eran Tauber, Department of Genetics, Leicester University
Lab 227, Department of Infection, Immunity and Inflammation, Leicester University
Phenotypes of 26 Jax-mouse strains
Phenotypes of 26 Jax-mouse strains