trinity college dublin kari-trc shirakawa institute of animal genetics functional genomics to...

Trinity College Dublin

KARI-TRC

Shirakawa Institute of Animal Genetics

Functional genomics to explore host response to trypanosome infection in particular and

stress in general.

Trinity College Dublin

KARI-TRC

Shirakawa Institute of Animal Genetics

Trypanosomosis

Is a fatal disease of livestock.The livestock equivalent

of sleeping sickness in humans

T. congolense, T. vivax

T brucei rhodesiense T gambiense

Role of livestock -

(should we all be vegetarians?)

Ghibe valley, Ethiopia

QuickTime™ and aDV - PAL decompressor

are needed to see this picture.

Bovins

Bovins et GlossinesGlossines

CattleTsetseCattle and tsetse

Origins of N’Dama and Boran cattle

N’DamaBoran

0 15 30 45 60 75 90 105 120 135 15030

50

70

90

110

N’DamaBoran

% change in PCV

days after infection

Studying the tolerant/susceptible phenotype has problems:

• Separating cause from effect

• Separating relevant from irrelevant.

• Dominance of the ‘what is happening to this weeks trendy gene/protein/cytokine?’ approach.

Studying tolerance susceptibility any way has problems:

• We have no idea of the mechanisms

• We have no idea of effector tissue

•We have no in vitro model

‘Mapping trypanotolerance loci of the N’Dama would allow

the rapid introgression of desired traits from other breeds

into the N’Dama, while retaining the trypanotolerance

traits; or the rapid introgression of the trypanotolerance

trait from the N’Dama to other breeds. Mapping of

trypanotolerance loci would also be the first step in their

eventual cloning and manipulation through genetic

engineering techniques’

M. Soller and J.S. Beckmann, M. Soller and J.S. Beckmann, FAO Consultation Report, March 1987FAO Consultation Report, March 1987

CCER June 2002

Contribution of 10 genes from Boranand N’Dama

cattle to reduction in degree of trypanosomosisBoran (relatively susceptible)

The N’Dama and Boran each contribute trypanotolerance alleles at 5 of the 10 most significant QTL, indicating that a synthetic breed could

have even higher tolerance than the N’Dama.

N’Dama (tolerant)

-15-10-505

1015

-15-10-50

51015

MMU1

MMU17

MMU5

D17Mit16

D17Mit46

D17Mit7 D5Mit233

D5Mit24

D5Mit114

D1Mit102

D1Mit403

D1Nds2

D1Mit113

0

120cM

80

40

In mice, we mapped three genomic regions which determine survival time following T. congolense infection

•What are these genes ?

•How do they affect survival ?

•What response pathways are common to mouse/cow/human ?

•What does that tell us about how to survive trypanosome challenge ?

The mapping data gives us a point of attack for a functional approach.

Functional genomics technology allows us to look at

what genes respond to infection

And especially what genes respond differently to infection

Expression analysis of cow and mouse, resistant and

susceptible.

Expression studiesCow

N’dama vs Boran time courseN’dama x Boran backcross

Mouse C57 vs A/J & Balb/c time course C57*A/J congenics

Various mouse mutants(C57*A/J BAC transgenics)

Cattle Microarray Time Course Design

Cattle Microarray Time Course Design

Day 0 12 15 18 21 26 29 32 35

Boran 20 5 5 5 5 5 5 5 5N'dama 20 5 5 5 5 5 5 5 5

Boran 10 5 5N'dama 10 5 5

Boran 10 5 5N'dama 10 5 5

Numbers sampled at each day

Liver

Lymph Node

Spleen

Principle components analysis of data from genome-wide expression analysis comparing gene expression in liver of Ndama (red) vs Boran (blue) in response to infection with T. congolense. Light colour day 29 post infection, dark day 32 post infection. Components 1 and 2. (Components 3 and 4 separate by day post infection)

PCA Liver day 0. 1st component tissue, this is 2nd

Principle components analysis of data from genome-wide expression analysis comparing gene expression in spleen of Ndama (red) vs Boran (blue) in response to infection with T. congolense. Light colour day 29 post infection, dark day 32 post infection. Components 1 and 2. (Components 3 and 4 separate by day post infection)

And the same data for spleen.

The biggest effect we see (after tissue) is breed.

Analysis

• What genes are differentially expressed genomewide?

• What pathways are they members of?• What pathways involve genes in the

QTL?• What pathways are in both lists ?• Prioritise the list by 'degree of change'• Look at the biology of each network

Paraoxonase 3 Chr 4 Near Parasitaemia QTL??

Paraoxonase 3 (PON3)

• PON1 knockout mice are more susceptible to T. congolense• PON3 is a 40-kDa protein associated with the

high density lipoprotein fraction of serum• PON3 rapidly hydrolyzes lactones such as

statin prodrugs (e.g. lovastatin)• PON3 is more efficient than rabbit PON1 in

protecting low density lipoprotein from copper-induced oxidation

Microarray design at each time point

Resistant C57BL/6Susceptible AJ

Mouse time course. Liver.

Cholesterol metabolism

Endogenous cholesterol production increases after

infection

Total Cholesterol levelsCHOLESTEROL

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

0 8 21 4

DAYS POST INFECTION

AJ _HIGH_FAT

AJ _LOW_FAT

BALB_HIGH_FAT

BALB_LOW_FAT

c57_HIGH_FAT

C57_LOW_FAT

African cows to Salford ICUHigh cholesterol in African cattle identified as aprotective factor against death from trypanosomiasis

Is high cholesterol a protective factor inhumans undergoing extreme inflammation?

ICU data and physicians in Salford ‘lab’ accessible,plus heamoglobin, creatinine and glucose ‘clamping’from normal ICU practice minimises major confounders

Data cleaning, meta-data capture, analysis

1.5

1.7

1.9

2.1

2.3

2.5

2.7

2.9

3.1

3.3

3.5

0 1 2 3 4 5 6 7 8 9 10

Day from admission to ICU

Tota

l seru

m c

hole

ste

rol

Survived

Died

Congenics

• Lines fixed for alternative ‘alleles’ of each QTL on a susceptible background.

Genotype males and mate carriers to female A/J

Genotype males and select individualscarrying donor Tir haplotypes on Chr 1, 5 or 17.

Genotype males, select individualswith the shortest donor flanks

Genotype, select carriers and intercross

Genotype all progeny, select homozygotesfixed for alternative haplotypes and expand

Cross susceptible recipient (A/J) with Resistant (C57BL/6) donor

F1

BC1

BC4

A/J C57BL/6

TirnBC6

TirnBC7

TirnAA TirnCC

A/J

Breeding congenic Mice carrying the Trypanotolerance QTLs

Response of Congenic mice to T. congolense infection

0

20

40

60

80

100

120

0 - 10 11 - 20 21-30 31-40 41-50 51-60 61-70 71-80

Days post intection

% s

urv

ivin

g

Tir1AA (n = 20)Tir1CC (n = 25)Tir2AA (n = 60)Tir2CC (n = 120)Tir3AA (n = 20)Tir3CC (n = 90)TirnAA (n = 100)

A/J sequence C57A/J sequence C57

number of differentially expressed genes in C57 v A/J (green)

number of differentially expressed genes in Tir1AA v Tir1CC (black, *>0.99)

Cxcl1 - inflammatory response. Tir 2!

A/J v C57:

expression differences

(fold change >0.5, P <0.01)

sequence differences (number of informative SNPs in the 1Kb upstream of each probed gene) across

the C57 and A/J genomes. (summed in 50 probe bins)

What to do with candidate genes

• All the obvious things

• Plus

• Exploit the unique populations (and high density SNP panels) of cattle– ‘Recently’ admixed resistant * susceptible– Multiple resistant breeds– Multiple resistant species (sheep, goat, wildlife)

Some conclusions

Expression analysis in cow and mouse has revealed some unexpected pathways and interactions. (Survival seems to be about the innate immune response and managing cholesterol)

Overlaying QTL and expression data has been incredibly informative.

We have learned a lot about host response to trypanosomes, but also about:

How to survive a tryps infection

How to survive in an ICU in Salford

Fundamentals of genome regulation.

If you do high quality science there will be high quality - but unpredictable - outcomes.

Expression analysis in cow and mouse has revealed some unexpected pathways and interactions. (Survival seems to be about the innate immune response and managing cholesterol)

Overlaying QTL and expression data has been incredibly informative.

We have learned a lot about host response to trypanosomes, but also about:

How to survive a tryps infection

How to survive in an ICU in Salford

Fundamentals of genome regulation.

If you do high quality science there will be high quality - but unpredictable - outcomes.

How to analyse this type of data and extract ‘important’ differences.

Overlaying multiple species has been extremely valuable.

There is a clear synergy to be won and additional livestock species would be extremely valuable for a range of traits……

(the international sheep consortium needs to develop genetics and genomics resources - draft / skim genome sequence, dense coverage SNP panels etc.).

Trinity College Dublin

KARI-TRC

Shirakawa Institute of Animal Genetics

Intersection of Cholesterol and Inflammatory pathways

Dunn et al Journal of Experimental MedicineVol. 203, No. 2, February 20, 2006 401–412

Th2 bias

Suppression of cholesterol synthesis