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QTN modulating the transcription rate of a chromosome domain encompassing PLAG1 control bovine stature.

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 …

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QTN modulating the transcription rate of a chromosome domain encompassing PLAG1 control bovine stature.

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  1. QTN modulating the transcription rate of a chromosome domain encompassing PLAG1 control bovine stature. Michel Georges University of Liège Belgium

  2. 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”

  3. Acknowledgments • UAG / Liège • L. Karim • H. Takeda • L. Lin • T. Druet • F. Farnir • B. Grisart • N. Cambisano • W. Coppieters • Boviquest / NZ • J. Arias • S. Davis • B. Harris • M. Keehan • M. Littlejohn • R. Spelman

  4. Plan • Mapping the QTL • Stature • A QTL affecting stature maps to BTA14 • L+LD fine-mapping defines a 750 Kb CI • Genetic identification of the QTN • HT sequencing identifies 13 candidate pQTN • Exploiting haplotype diversity to eliminate 5/13 candidate pQTN • Functional analysis 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 • Naturally occurring null allele excludes CHCHD7 • Conclusions

  5. Stature

  6. Stature • Human: • Paradigmatic Quantitative Trait • h280% • Quasi-infinitesimal architecture • Dog: • 5 loci explain nearly all the difference of stature between breeds. • Cattle: • Auroch: 2m=> domestic cattle: 1.1-1.5m • Economically important trait • h225-80% • Many reported “QTL”

  7. Plan • Mapping the QTL • Stature • A QTL affecting stature maps to BTA14 • L+LD fine-mapping defines a 780 Kb CI • Genetic identification of the QTN • HT sequencing identifies 13 candidate pQTN • Exploiting haplotype diversity to eliminate 5/13 candidate pQTN • Functional analysis 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 • Naturally occurring null allele excludes CHCHD7 • Conclusions

  8. A QTL affecting stature maps to BTA14: HF x J F2 population 500 traits

  9. A QTL affecting stature maps to BTA14: line-cross model 294 microsatellites

  10. A QTL affecting stature maps to BTA14: ½-sib model Across-family analysis – 1 QTL 8->56 μsat.

  11. A QTL affecting stature maps to BTA14: ½-sib model Within family analysis – effects Within family analysis – significance

  12. Plan • Mapping the QTL • Stature • A QTL affecting stature maps to BTA14 • L+LD fine-mapping defines a 780 Kb CI • Genetic identification of the QTN • HT sequencing identifies 13 candidate pQTN • Exploiting haplotype diversity to eliminate 5/13 candidate pQTN • Functional analysis 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 • Naturally occurring null allele excludes CHCHD7 • Conclusions

  13. 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

  14. L+LD fine-mapping defines 780 Kb interval: outbred pop. Substitution effects of hidden haplotype states “Q” 1% of phenotypic variance “q” Multipoint analysis – 1 QTL/2 QTL 3% of phenotypic variance No unique haplotype associated with Q or q

  15. Plan • Mapping the QTL • Stature • A QTL affecting stature maps to BTA14 • L+LD fine-mapping defines a 780 Kb CI • Genetic identification of the QTN • HT sequencing identifies 13 candidate pQTN • Exploiting haplotype diversity to eliminate 5/13 candidate pQTN • Functional analysis 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 • Naturally occurring null allele excludes CHCHD7 • Conclusions

  16. 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

  17. 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.

  18. HT sequencing of 780 Kb interval: =>13 candidate QTN

  19. HT sequencing of 780 Kb interval: =>13 candidate QTN

  20. HT sequencing of 780 Kb interval: =>13 candidate QTN

  21. Plan • Mapping the QTL • Stature • A QTL affecting stature maps to BTA14 • L+LD fine-mapping defines a 780 Kb CI • Genetic identification of the QTN • HT sequencing identifies 13 candidate pQTN • Exploiting haplotype diversity to eliminate 5/13 candidate pQTN • Functional analysis 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 • Naturally occurring null allele excludes CHCHD7 • Conclusions

  22. Across breed haplotype diversity => 8 candidate QTN

  23. Across breed haplotype diversity => 8 candidate QTN

  24. Across breed haplotype diversity => 8 candidate QTN

  25. Plan • Mapping the QTL • Stature • A QTL affecting stature maps to BTA14 • L+LD fine-mapping defines a 780 Kb CI • Genetic identification of the QTN • HT sequencing identifies 13 candidate pQTN • Exploiting haplotype diversity to eliminate 5/13 candidate pQTN • Functional analysis 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 • Naturally occurring null allele excludes CHCHD7 • Conclusions

  26. Intact ORFs support regulatory pQTN

  27. Plan • Mapping the QTL • Stature • A QTL affecting stature maps to BTA14 • L+LD fine-mapping defines a 780 Kb CI • Genetic identification of the QTN • HT sequencing identifies 13 candidate pQTN • Exploiting haplotype diversity to eliminate 5/13 candidate pQTN • Functional analysis 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 • Naturally occurring null allele excludes CHCHD7 • Conclusions

  28. Expression analysis: M&M • 79 fetuses • QRT-PCR (SYBR and/or 3’exonucl.) • x/8 internal controls selected with geNorm • ≤ 4 amplicons/gene

  29. The pQTN affect expression of all genes in conserved domain

  30. The pQTN affect expression of all genes in conserved domain Average: 20.86 ≈ 1.8

  31. Allelic imbalance at (pre-)mRNA level => transcriptional effect

  32. Conservation of synteny suggests domain “regulon”

  33. Plan • Mapping the QTL • Stature • A QTL affecting stature maps to BTA14 • L+LD fine-mapping defines a 780 Kb CI • Genetic identification of the QTN • HT sequencing identifies 13 candidate pQTN • Exploiting haplotype diversity to eliminate 5/13 candidate pQTN • Functional analysis 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 • Naturally occurring null allele excludes CHCHD7 • Conclusions

  34. 3/8 candidate pQTN affect Phastcons elements

  35. Reporter assays and EMSA support 2 promotor pQTN * *

  36. Reporter assays and EMSA support 2 promotor pQTN

  37. Reporter assays and EMSA support 2 promotor pQTN

  38. Reporter assays and EMSA support 2 promotor pQTN

  39. Plan • Mapping the QTL • Stature • A QTL affecting stature maps to BTA14 • L+LD fine-mapping defines a 780 Kb CI • Genetic identification of the QTN • HT sequencing identifies 13 candidate pQTN • Exploiting haplotype diversity to eliminate 5/13 candidate pQTN • Functional analysis 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 • Naturally occurring null allele excludes CHCHD7 • Conclusions

  40. Pick your favorite gene … X X

  41. Pick your favorite gene … X X

  42. Formal test for gene causality:Distribution of rare variant Σ=5% Σ=17%

  43. Formal test for gene causality:reciprocal hemizygosity Steinmetz et al. 2002

  44. Formal test for gene causality: quantitative complementation

  45. Naturally occurring null allele excludes CHCHD7 CHCHD7 cis-eQTLwith distinct segregation vector (vs “pQTL”)

  46. Naturally occurring null allele excludes CHCHD7 “eQTN” is a donor splice site variant

  47. Naturally occurring null allele excludes CHCHD7

  48. 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

  49. No failure to quantitatively complement

  50. 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

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