Transcript Introductory genetics for veterinary students

QTN modulating the transcription
rate of a chromosome domain
encompassing PLAG1 control bovine
stature.
Michel Georges
University of Liège
Belgium
Introduction
 GWAS identify …
 … risk loci
 150 Kb
 3.5 genes (range: 0-35)
 … but neither genes, nor causal variants
 Genomic selection …
 … is effective
 … has confirmed quasi-infinitesimal
component for most traits
 … is a new “black box”
Acknowledgments
 UAG / Liège
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L. Karim
H. Takeda
L. Lin
T. Druet
F. Farnir
B. Grisart
N. Cambisano
W. Coppieters
 Boviquest / NZ
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J. Arias
S. Davis
B. Harris
M. Keehan
M. Littlejohn
R. Spelman
Plan
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Mapping the QTL
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Genetic identification of the QTN
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Intact ORFs support regulatory pQTN
pQTN affects expression of PLAG1-encompassing domain
Reporter assays and EMSA support causality of pQTN in PLAG1-CHCHD7
bidirectional promoter
Identifying the causative gene
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HT sequencing identifies 13 candidate pQTN
Exploiting haplotype diversity to eliminate 5/13 candidate pQTN
Functional analysis of the QTN
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Stature
A QTL affecting stature maps to BTA14
L+LD fine-mapping defines a 750 Kb CI
Naturally occurring null allele excludes CHCHD7
Conclusions
Stature
Stature
 Human:
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Paradigmatic Quantitative Trait
h2 80%
Quasi-infinitesimal architecture
 Dog:
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5 loci explain nearly all the difference of stature between
breeds.
Cattle:
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Auroch: 2m=> domestic cattle: 1.1-1.5m
Economically important trait
h2 25-80%
Many reported “QTL”
Plan
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Mapping the QTL
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Genetic identification of the QTN
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Intact ORFs support regulatory pQTN
pQTN affects expression of PLAG1-encompassing domain
Reporter assays and EMSA support causality of pQTN in PLAG1-CHCHD7
bidirectional promoter
Identifying the causative gene
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
HT sequencing identifies 13 candidate pQTN
Exploiting haplotype diversity to eliminate 5/13 candidate pQTN
Functional analysis of the QTN
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Stature
A QTL affecting stature maps to BTA14
L+LD fine-mapping defines a 780 Kb CI
Naturally occurring null allele excludes CHCHD7
Conclusions
A QTL affecting stature maps to
BTA14: HF x J F2 population
500 traits
A QTL affecting stature maps to
BTA14: line-cross model
294 microsatellites
A QTL affecting stature maps to
BTA14: ½-sib model
Across-family analysis – 1 QTL
8->56 μsat.
A QTL affecting stature maps to
BTA14: ½-sib model
Within family analysis – effects
Within family analysis – significance
Plan

Mapping the QTL
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Genetic identification of the QTN
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Intact ORFs support regulatory pQTN
pQTN affects expression of PLAG1-encompassing domain
Reporter assays and EMSA support causality of pQTN in PLAG1-CHCHD7
bidirectional promoter
Identifying the causative gene


HT sequencing identifies 13 candidate pQTN
Exploiting haplotype diversity to eliminate 5/13 candidate pQTN
Functional analysis of the QTN
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Stature
A QTL affecting stature maps to BTA14
L+LD fine-mapping defines a 780 Kb CI
Naturally occurring null allele excludes CHCHD7
Conclusions
L+LD fine-mapping defines
780 Kb interval: F2 population
• + 925 SNPs
• LD  non inbred F0
Multipoint analysis – 1 QTL/2 QTL
Single-point analysis – 1 QTL
10% of phenotypic variance
- Mixed model including “animal effect”
- Hidden Haplotype States
L+LD fine-mapping defines
780 Kb interval: outbred pop.
Substitution effects of
hidden haplotype states
1% of phenotypic variance
Multipoint analysis – 1 QTL/2 QTL
No unique haplotype associated
with Q or q
“q”
3% of phenotypic variance
“Q”
Plan

Mapping the QTL
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

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Genetic identification of the QTN
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
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Intact ORFs support regulatory pQTN
pQTN affects expression of PLAG1-encompassing domain
Reporter assays and EMSA support causality of pQTN in PLAG1-CHCHD7
bidirectional promoter
Identifying the causative gene


HT sequencing identifies 13 candidate pQTN
Exploiting haplotype diversity to eliminate 5/13 candidate pQTN
Functional analysis of the QTN
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


Stature
A QTL affecting stature maps to BTA14
L+LD fine-mapping defines a 780 Kb CI
Naturally occurring null allele excludes CHCHD7
Conclusions
HT sequencing of 780 Kb
interval: =>13 candidate QTN
 M&M:
 “Progeny-tested” chromosomes
of six F1 sires
 103 long range PCR products
 Sire-specific multiplex identifiers
(MIDs)
 Roche FLX
MASA: Converting a polygenic trait in a series of monogenic entities
HT sequencing of 780 Kb
interval: =>13 candidate QTN
 Results:
 Average 20-fold coverage / sire
 9,572 variants  π: 1/300
 14 candidate QTN  segregation
pattern compatible with QTL
genotype.
HT sequencing of 780 Kb
interval: =>13 candidate QTN
HT sequencing of 780 Kb
interval: =>13 candidate QTN
HT sequencing of 780 Kb
interval: =>13 candidate QTN
Plan

Mapping the QTL




Genetic identification of the QTN



Intact ORFs support regulatory pQTN
pQTN affects expression of PLAG1-encompassing domain
Reporter assays and EMSA support causality of pQTN in PLAG1-CHCHD7
bidirectional promoter
Identifying the causative gene


HT sequencing identifies 13 candidate pQTN
Exploiting haplotype diversity to eliminate 5/13 candidate pQTN
Functional analysis of the QTN




Stature
A QTL affecting stature maps to BTA14
L+LD fine-mapping defines a 780 Kb CI
Naturally occurring null allele excludes CHCHD7
Conclusions
Across breed haplotype
diversity => 8 candidate QTN
Across breed haplotype
diversity => 8 candidate QTN
Across breed haplotype
diversity => 8 candidate QTN
Plan

Mapping the QTL




Genetic identification of the QTN



Intact ORFs support regulatory pQTN
pQTN affects expression of PLAG1-encompassing domain
Reporter assays and EMSA support causality of pQTN in PLAG1-CHCHD7
bidirectional promoter
Identifying the causative gene


HT sequencing identifies 13 candidate pQTN
Exploiting haplotype diversity to eliminate 5/13 candidate pQTN
Functional analysis of the QTN




Stature
A QTL affecting stature maps to BTA14
L+LD fine-mapping defines a 780 Kb CI
Naturally occurring null allele excludes CHCHD7
Conclusions
Intact ORFs support
regulatory pQTN
Plan

Mapping the QTL
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


Genetic identification of the QTN



Intact ORFs support regulatory pQTN
pQTN affects expression of PLAG1-encompassing domain
Reporter assays and EMSA support causality of pQTN in PLAG1-CHCHD7
bidirectional promoter
Identifying the causative gene


HT sequencing identifies 13 candidate pQTN
Exploiting haplotype diversity to eliminate 5/13 candidate pQTN
Functional analysis of the QTN




Stature
A QTL affecting stature maps to BTA14
L+LD fine-mapping defines a 780 Kb CI
Naturally occurring null allele excludes CHCHD7
Conclusions
Expression analysis: M&M
 79 fetuses
 QRT-PCR (SYBR and/or 3’exonucl.)
 x/8 internal controls selected with
geNorm
 ≤ 4 amplicons/gene
The pQTN affect expression of
all genes in conserved domain
The pQTN affect expression of
all genes in conserved domain
Average: 20.86 ≈ 1.8
Allelic imbalance at (pre-)mRNA
level => transcriptional effect
Conservation of synteny
suggests domain “regulon”
Plan

Mapping the QTL




Genetic identification of the QTN



Intact ORFs support regulatory pQTN
pQTN affects expression of PLAG1-encompassing domain
Reporter assays and EMSA support causality of pQTN in PLAG1-CHCHD7
bidirectional promoter
Identifying the causative gene


HT sequencing identifies 13 candidate pQTN
Exploiting haplotype diversity to eliminate 5/13 candidate pQTN
Functional analysis of the QTN




Stature
A QTL affecting stature maps to BTA14
L+LD fine-mapping defines a 780 Kb CI
Naturally occurring null allele excludes CHCHD7
Conclusions
3/8 candidate pQTN affect
Phastcons elements
Reporter assays and EMSA
support 2 promotor pQTN
**
Reporter assays and EMSA
support 2 promotor pQTN
Reporter assays and EMSA
support 2 promotor pQTN
Reporter assays and EMSA
support 2 promotor pQTN
Plan

Mapping the QTL




Genetic identification of the QTN



Intact ORFs support regulatory pQTN
pQTN affects expression of PLAG1-encompassing domain
Reporter assays and EMSA support causality of pQTN in PLAG1-CHCHD7
bidirectional promoter
Identifying the causative gene


HT sequencing identifies 13 candidate pQTN
Exploiting haplotype diversity to eliminate 5/13 candidate pQTN
Functional analysis of the QTN




Stature
A QTL affecting stature maps to BTA14
L+LD fine-mapping defines a 780 Kb CI
Naturally occurring null allele excludes CHCHD7
Conclusions
Pick your favorite gene …
X
X
Pick your favorite gene …
X
X
Formal test for gene causality:
Distribution of rare variant
Σ=5%
Σ=17%
Formal test for gene causality:
reciprocal hemizygosity
Steinmetz et al. 2002
Formal test for gene causality:
quantitative complementation
Naturally occurring null allele
excludes CHCHD7
CHCHD7 cis-eQTLwith distinct
segregation vector (vs “pQTL”)
Naturally occurring null allele
excludes CHCHD7
“eQTN” is a donor
splice site variant
Naturally occurring null allele
excludes CHCHD7
Naturally occurring null allele
excludes CHCHD7
 Splice site variant affects transcript levels in
multiple (all?) tissues
 pQTL and eQTL have different segregation
vector
 pQTL effect on stature is same for 4
segregating sires
 eQTN has no significant “residual” effect on
stature
 No failure to quantitatively complement
No failure to quantitatively
complement
Naturally occurring null allele
excludes CHCHD7
 Splice site variant affects transcript levels in multiple
(all?) tissues
 pQTL and eQTL have different segregation vector
 pQTL effect on stature is same for 4 segregating sires
 eQTN has nosignificant “residual” effect on stature
 No failure to quantitatively complement
=> CHCHD7 can not be sole causative
gene
Plan

Mapping the QTL




Genetic identification of the QTN



Intact ORFs support regulatory pQTN
pQTN affects expression of PLAG1-encompassing domain
Reporter assays and EMSA support causality of pQTN in PLAG1-CHCHD7
bidirectional promoter
Identifying the causative gene


HT sequencing identifies 13 candidate pQTN
Exploiting haplotype diversity to eliminate 5/13 candidate pQTN
Functional analysis of the QTN




Stature
A QTL affecting stature maps to BTA14
L+LD fine-mapping defines a 750 Kb CI
Naturally occurring null allele excludes CHCHD7
Conclusions
Conclusions
 QTN modulating the transcription rate of a chromosome
domain encompassing PLAG1 control bovine stature
 Domestic animal populations have unique features
facilitating the genetic dissection of complex traits
(line-crosses, harems, reduced effective population size,
haplotype diversity)
 Haplotype sharing may not always be effective for the
identification of old QTN
 The QCA can be applied in outbred populations using
naturally occurring null alleles
Thank you for your attention
…