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