Combine GX and QTL analysis - University of Leicester · 19 29.3 Lipk lipase, family member K 19...

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

02/10/2012

2

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


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


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

02/10/2012

3


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 29.79 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

02/10/2012

4

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

02/10/2012

5

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 29.74 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 29.79 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

02/10/2012

6

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



Combine GX and QTL analysis - University of Leicester · 19 29.3 Lipk lipase, family member K 19...

Documents

Transcript of Combine GX and QTL analysis - University of Leicester · 19 29.3 Lipk lipase, family member K 19...