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Discovery of a putative causal mutation for Angus dwarfism

Discovery of a putative causal mutation for Angus dwarfism. Summary of phenotypic characteristics. Shortened limbs, but normal body size Decreased long bone and vertebral length Incorrect endochondral ossification Premature calcification of chondrocytes Disorganized arrangement of osteocytes

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Discovery of a putative causal mutation for Angus dwarfism

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  1. Discovery of a putative causal mutation for Angus dwarfism

  2. Summary of phenotypic characteristics • Shortened limbs, but normal body size • Decreased long bone and vertebral length • Incorrect endochondral ossification • Premature calcification of chondrocytes • Disorganized arrangement of osteocytes • Abnormal osteocyte phenotype

  3. Calves at 7 months vs. birth Two bulls at Birth 7 Months 16cM 18cM Normal Dwarf C/T T/T T/T Normal Dwarf female Dwarf male

  4. Strategy for finding the gene mutation for dwarfism • Consider known bovine dwarfisms • Select candidate bovine chromosomes for linkage analysis • Find a suitable population for linkage analysis

  5. Family Structure Sire 1 Sire 2 Sire 3 Dam 4 Dam 6 Sire 5 Dam 7 Dam 8 Dam 9 Sire 16 Dam 14 Sire 15 Sire 10 Dam 11 Dam 12 Dam 13 Dwarf2 Dwarf6 Dwarf1 Dwarf3 Dwarf4/5

  6. Pedigree Analysis

  7. Are known bovine mutations responsible Angus dwarfism? 1356 TTTTCAAAGG C GAGCTGAAGC - Normal limbin TTTTCAAAGG TGAGCTGAAGC - Mutant limbin TTTTCAAAGG C GAGCTGAAGC - Angus dwarf/normal 2054/2055 AGGGACCTGG CA GCCAGGGCCG - Normal limbin AGGGACCTGG G GCCAGGGCCG - Mutantlimbin AGGGACCTGG CAGCCAGGGCCG – Angus dwarf/normal Mishra et al., 2003

  8. Are known bovine mutations responsible Angus dwarfism?

  9. Whole Genome Scan • Systematically evaluate the bovine genome • Chromosomes known to contain genes implicated in dwarfism in other species were given a priority for evaluation • Evenly spaced microsatellites • Genotyping done in Dr. Georges lab in Belgium

  10. Pinpointing the genomic region associated with dwarfism • Tested marker intervals for Likelihood of odds (LOD) with dwarfism LOD =Log10 Marker Interval contains dwarf mutation Dwarf mutation is on a different chromosome • Critical value: LOD=3.0 • Problem: missing genotypes

  11. Initial Results of Linkage mapping

  12. BTA 6 BTA 6 Marker 1 40 cM Improved Marker Map Coverage Interval 8 Known dwarf Gene = Limbin

  13. Summary of initial results • Known dwarf mutations are not responsible for Angus dwarfism • Linkage detected to BTA6 • Limbin eliminated as a candidate gene • Critical region refined to 2.8 cM

  14. Cow with known relatives with dwarf progeny and same haplotype 1 1 1 1 2 2 1 1 X = dwarf carrier 1 1 1 1 2 1 2 1 = normal Prob (dwarf carrier/ relatives phenotype, genotype, individuals genotype) Former solution, probabilities

  15. Candidate genes from conserved syntenic blocks across human, mouse and rat contain HEL308 PLAC8 HPSE COPS4 THAP9 SCD4 MASA HNRPD PRKG2 BMP3 FGF5 PRDM8 ANTXR2 GDEP GK2 PAQR3 BMP2K ANXA3 FRAS1 MRPL1 HSA4 MMU5 RN14 BTA

  16. Selection of candidate genes • Gene Function • Knockout analysis in the mouse • Naturally occurring mutants • Based on these criteria, 4 genes were selected

  17. Overview of positional candidate genes • Bone Morphogenetic Protein 2 Kinase (BMP2K) • Fibroblast Growth Factor 5 (FGF5) • Bone Morphogenetic Protein 3 • cGMP-dependant, type II, protein kinase (PRKG2)

  18. 1 2 3 4 5 6 7 8 9 10 100 75 40 330 3 1 14 47 12 3 98 (kB) Candidate genes genotyped

  19. Interval mapping Marker Interval

  20. Wild-type/ Unaffected T R R P Carrier T R R/Z P Dwarf/ affected T R Z A mutation in PRKG2 is likely causal PRKG2=cGMP dependant, type II, protein kinase.

  21. Mutant Wild-type NTP binding domain Protein kinase domain Α-helix domain 80 240 480 762AA Exon15 C/T Predicted PRKG2 in dwarves 678AA The PRKG2 mutation drastically changes protein structure B. A.

  22. PRKG2 is necessary for long bone growth Femur Transplants Long Bone Length Growth Plate Wildtype Knockout Pfeifer et al. 1996

  23. Additional functional work Female dwarf Male dwarf Male carrier

  24. Test for Angus dwarfism Shoulder Height in Inches

  25. N=4 reps/ trt Dwarf vs. control (P=0.0028) WT vs. dwarf (P=0.0028) WT vs. control (P=0.39)

  26. Evidence supporting causality of PRKG2 mutation • PRKG2 knockout mice result in dwarfism • A natural deletion in PRKG2 causes rat dwarfism • PRKG2 signaling alters SOX9 function, which is required to allow chondrocyte maturation. • Stop codon is introduced in PRKG2’s kinase domain • The mutation is 100% concordant with dwarves, carriers, and wild-type phenotypes

  27. Acknowledgements Iowa State James Koltes Bishnu MishraUniversity of Nebraska Dr. Zhiliang Hu David Steffen University of LiegeUniversity of Sydney Michel Georges Julie Cavanagh Wouter Coppieters Imke Tammen Haruko Takeda Ohio State University Washington State UniversityChuck Hines Rowland Cobbold American Angus Association Tawfik Aboellail Bryce Schumann

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