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Positional identification of a regulatory mutation in the porcine IGF2 gene having a major QTL effect on muscle mass.

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Presentation on theme: "Positional identification of a regulatory mutation in the porcine IGF2 gene having a major QTL effect on muscle mass."— Presentation transcript:

1 Positional identification of a regulatory mutation in the porcine IGF2 gene having a major QTL effect on muscle mass.

2 M. Georges et al. ; IWMGQSG Acknowledgments ULg: ULg: Minh Nguyen Minh Nguyen Carine Nezer Carine Nezer Catherine Collette Catherine Collette Laurence Moreau Laurence Moreau Michel Georges Michel Georges Gentec: Gentec: Nadine Buys Nadine Buys Uppsala: Anne-Sophie Van Laere Martin Braunschweig Michael Tally Valérie Amarger Goran Andersson Leif Andersson Roslin: Chris Haley Alan Archibald

3 M. Georges et al. ; IWMGQSG Molecular dissection of complex traits The majority of medically and agronomically important phenotypes are continuously distributed multifactorial traits. The majority of medically and agronomically important phenotypes are continuously distributed multifactorial traits. The nature of quantitative-trait variation is one of the last unexplored frontiers in genetics, awaiting the future cloning and definitive identification of quantitative-trait determinants, whether they be genetic or epigenetic. Rutherford and Henikoff (2003) The nature of quantitative-trait variation is one of the last unexplored frontiers in genetics, awaiting the future cloning and definitive identification of quantitative-trait determinants, whether they be genetic or epigenetic. Rutherford and Henikoff (2003)

4 M. Georges et al. ; IWMGQSG Livestock populations offer unique advantages for complex trait analysis A growing list of phenotypes is being systematically recorded on a substantial proportion of the breeding population. A growing list of phenotypes is being systematically recorded on a substantial proportion of the breeding population. Individual phenotypes are being processed to compute environmental and genetic variance components, from which estimates of heritability can be derived. Individual phenotypes are being processed to compute environmental and genetic variance components, from which estimates of heritability can be derived. Extant pedigrees with a suitable structure for the mapping of QTL underlying the within-population genetic variance can often be readily collected. Extant pedigrees with a suitable structure for the mapping of QTL underlying the within-population genetic variance can often be readily collected.

5 M. Georges et al. ; IWMGQSG Livestock populations offer unique advantages for complex trait analysis The effective population size is typically restricted: The effective population size is typically restricted: The genetic complexity of the trait in terms of allelic and locus heterogeneity is bound to be reduced. The genetic complexity of the trait in terms of allelic and locus heterogeneity is bound to be reduced. Linkage disequilibrium (LD) in livestock extends over tens of centimorgans. Linkage disequilibrium (LD) in livestock extends over tens of centimorgans. Livestock populations now resemble advanced inter-cross lines between highly divergent genomes exhibiting nucleotide diversities as high as 1/200. Livestock populations now resemble advanced inter-cross lines between highly divergent genomes exhibiting nucleotide diversities as high as 1/200. Experimental livestock inter- and back-cross pedigrees can be generated at will to map QTL that underlie the between population genetic variance. Experimental livestock inter- and back-cross pedigrees can be generated at will to map QTL that underlie the between population genetic variance.

6 I. An imprinted QTL with major effect on muscle mass maps to the porcine IGF2 locus.

7 M. Georges et al. ; IWMGQSG A QTL with major effect on muscle mass and fat deposition maps to distal SSC2p Piétrain x Large White F2

8 M. Georges et al. ; IWMGQSG SSC2p is orthologous to HSA11pter-q13 Igf2 MyoD1

9 M. Georges et al. ; IWMGQSG The QTL co-localizes with IGF2

10 M. Georges et al. ; IWMGQSG IGF2 is imprinted in the pig

11 M. Georges et al. ; IWMGQSG The QTL is imprinted and expressed exclusively from the paternal allele

12 M. Georges et al. ; IWMGQSG The Piétrain & Large White IGF2 coding sequences are identical Large White x Piétrain F2 (Nezer et al., 1999) Large White x Piétrain F2 (Nezer et al., 1999) Large White x Wild Boar F2 (Jeon et al., 1999) Large White x Wild Boar F2 (Jeon et al., 1999) Large White x Meishan F2 (De Koning et al., 2000) Large White x Meishan F2 (De Koning et al., 2000) Large White x Berkshire F2 (Thomsen et al., 2002) Large White x Berkshire F2 (Thomsen et al., 2002) Similar results were obtained by others …

13 II. Haplotype sharing refines the QTL location to a 250 Kb segment containing the IGF2 gene

14 M. Georges et al. ; IWMGQSG Principle underlying proposed approach Q q

15 M. Georges et al. ; IWMGQSG QTL genotyping by « MASA » Identify large paternal half-sib pedigrees (Piétrain x Large White cross-bred boars) Identify large paternal half-sib pedigrees (Piétrain x Large White cross-bred boars) Genotype for distal SSC2p microsatellites Genotype for distal SSC2p microsatellites Compute z = log(H1/H0) assuming a = 2% Compute z = log(H1/H0) assuming a = 2% « MASA »: « MASA »: If z > 2, sire = « Qq » If z > 2, sire = « Qq » If z < 2, sire = « QQ » or « qq » If z < 2, sire = « QQ » or « qq »

16 M. Georges et al. ; IWMGQSG

17 Generate a denser map spanning the HSA 11p imprinted domain Develop CATS Develop CATS Identify SNPs Identify SNPs Screen BAC library Screen BAC library BAC-end sequences BAC-end sequences 51 SNPs (9 multisite haplotypes) 11 micros

18 M. Georges et al. ; IWMGQSG Assemble pools of « Q » and « q » chromosomes

19 M. Georges et al. ; IWMGQSG All "Q" chromosomes share a 250 Kb common haplotype encompassing the INS and IGF2 genes not present in the "q" chromosomes

20 M. Georges et al. ; IWMGQSG All "Q" chromosomes share a 250 Kb common haplotype encompassing the INS and IGF2 genes not present in the "q" chromosomes

21 III. Positional identification of a regulatory mutation in IGF2 causing a major QTL effect on muscle development in the pig. A. « Genetic » analysis

22 M. Georges et al. ; IWMGQSG Comparative sequence analysis of the INS-IGF2-H19 gene cluster identifies 97 evolutionary footprints (Amarger et al., 2002)

23 M. Georges et al. ; IWMGQSG Resequencing 3 Q and 8 q chromosomes for 28.5 Kb spanning INS-IGF2 identifies 33 putative QTN

24 M. Georges et al. ; IWMGQSG Resequencing 3 Q and 8 q chromosomes for 28.5 Kb spanning INS-IGF2 identifies 33 putative QTN « Q »: 1 clade, « q »: 2 clades « Q »: 1 clade, « q »: 2 clades 253 SNPs !! (1/186) 253 SNPs !! (1/186) 33 candidate QTN (2 in evolutionary footprint) 33 candidate QTN (2 in evolutionary footprint)

25 M. Georges et al. ; IWMGQSG Resequencing a heterozygous, non-segregating Hampshire sire identifies a recombination excluding TH-IGF2(I1) (- 9 candidate QTN)

26 M. Georges et al. ; IWMGQSG Resequencing a heterozygous, non-segregating Large White x Meishan sire identifies the QTN

27 M. Georges et al. ; IWMGQSG Resequencing a heterozygous, non-segregating Large White x Meishan sire identifies the QTN Meishan chromosome belongs to « Q » clade, from which it differs at one of the two candidate QTN in an evolutionary conserved footprint. Meishan chromosome belongs to « Q » clade, from which it differs at one of the two candidate QTN in an evolutionary conserved footprint. Large White chromosome belongs to « q » clade, and is identical to all « q »s at all 33 candidate QTN. Large White chromosome belongs to « q » clade, and is identical to all « q »s at all 33 candidate QTN. « MASA » in Edinburgh, confirms « q » status of Meishan chromosome. « MASA » in Edinburgh, confirms « q » status of Meishan chromosome. « Identifies the QTN !! » « Identifies the QTN !! »

28 M. Georges et al. ; IWMGQSG

29 III. Positional identification of a regulatory mutation in IGF2 causing a major QTL effect on muscle development in the pig. B. « Functional » analysis

30 M. Georges et al. ; IWMGQSG Background information Expected effect of the QTN: Expected effect of the QTN: « Increase IGF2 expression levels without altering the imprinting status. » Information from the mouse: Information from the mouse: « QTN is part of a region that contains a mesodermal silencer binding GCF2 when unmethylated (maternal allele) » « QTN is part of a region that contains a mesodermal silencer binding GCF2 when unmethylated (maternal allele) »

31 M. Georges et al. ; IWMGQSG In vivo analysis of the QTN effect The QTN does not affect imprinting The QTN does not affect imprinting

32 M. Georges et al. ; IWMGQSG In vivo analysis of the QTN effect A 3-fold increase of postnatal IGF2 mRNA expression is observed in skeletal muscle in (Q/Q or Q pat /q mat ) versus (q pat /Q mat or q/q). A 3-fold increase of postnatal IGF2 mRNA expression is observed in skeletal muscle in (Q/Q or Q pat /q mat ) versus (q pat /Q mat or q/q). Northern blot RT-PCR

33 M. Georges et al. ; IWMGQSG In vivo analysis of the QTN effect The QTN is located in a novel DMR. The QTN is located in a novel DMR. Methytlation is not affected by genotype

34 M. Georges et al. ; IWMGQSG In vitro analysis of the QTN effect « EMSA »: « EMSA »: The QTN abrogates binding of a nuclear factor present in C2C12 myoblasts. The QTN abrogates binding of a nuclear factor present in C2C12 myoblasts.

35 M. Georges et al. ; IWMGQSG In vitro analysis of the QTN effect « Reporter assay »: « Reporter assay »: In C2C12 cells, the « Q » allele increases P3-driven luciferase expression by 3.5 when compared to « q » In C2C12 cells, the « Q » allele increases P3-driven luciferase expression by 3.5 when compared to « q »

36 IV. Conclusions

37 M. Georges et al. ; IWMGQSG Conclusions We herein report the first identification of a regulatory point mutation underlying a QTL. We herein report the first identification of a regulatory point mutation underlying a QTL. This work illustrates the extraordinary resolution that is achievable in the molecular dissection of complex traits when using domestic animal resources. This work illustrates the extraordinary resolution that is achievable in the molecular dissection of complex traits when using domestic animal resources.

38 M. Georges et al. ; IWMGQSG Conclusions This work demonstrates that IGF2 has an important role in regulating mostnatal myogenesis, This work demonstrates that IGF2 has an important role in regulating mostnatal myogenesis, … and opens new avenues for modulating muscle development by targetting the identified sequence and the binding trans- acting factor. … and opens new avenues for modulating muscle development by targetting the identified sequence and the binding trans- acting factor.

39 M. Georges et al. ; IWMGQSG Thank you for your attention


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