Department of Animal Science Nov. 2006NBCEC Brown Bagger
Discovery of a putative causal mutation for Angus dwarfism
Department of Animal Science Nov. 2006NBCEC Brown Bagger
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
Department of Animal Science Nov. 2006NBCEC Brown Bagger
Calves at 7 months vs. birth
16cM 18cM
T/TT/TC/T
Normal Dwarffemale
Dwarfmale
7 Months
Two bulls at Birth
NormalDwarf
Department of Animal Science Nov. 2006NBCEC Brown Bagger
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
Department of Animal Science Nov. 2006NBCEC Brown Bagger
Dwarf1Dwarf1 Dwarf2Dwarf2 Dwarf3Dwarf3 Dwarf4/5Dwarf4/5 Dwarf6Dwarf6
Sire 10Sire 10 Dam 11Dam 11 Dam 12Dam 12 Dam 13Dam 13 Dam 14Dam 14 Sire 15Sire 15 Sire 16Sire 16
Sire 5Sire 5 Dam 6Dam 6 Dam 7Dam 7 Dam 8Dam 8 Dam 9Dam 9
Sire 1Sire 1 Sire 2Sire 2 Sire 3Sire 3 Dam 4Dam 4
Family Structure
Department of Animal Science Nov. 2006NBCEC Brown Bagger
Pedigree Analysis
Department of Animal Science Nov. 2006NBCEC Brown Bagger
Are known bovine mutations responsible Angus dwarfism?
1356TTTTCAAAGG C C GAGCTGAAGC - Normal limbinTTTTCAAAGG TT GAGCTGAAGC - Mutant limbinTTTTCAAAGG C C GAGCTGAAGC - Angus dwarf/normal
2054/2055AGGGACCTGG CA CA GCCAGGGCCG - Normal limbinAGGGACCTGG G G GCCAGGGCCG - Mutant limbinAGGGACCTGG CACA GCCAGGGCCG – Angus dwarf/normal
Mishra et al., 2003
Department of Animal Science Nov. 2006NBCEC Brown Bagger
Are known bovine mutations responsible Angus dwarfism?
Department of Animal Science Nov. 2006NBCEC Brown Bagger
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
Department of Animal Science Nov. 2006NBCEC Brown Bagger
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
Department of Animal Science Nov. 2006NBCEC Brown Bagger
Initial Results of Linkage mapping
Department of Animal Science Nov. 2006NBCEC Brown Bagger
Improved Marker Map Coverage
Marker 1
BTA 6
BTA 6
40 cM
Known dwarf Gene = Limbin
Interval 8
Department of Animal Science Nov. 2006NBCEC Brown Bagger
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
Department of Animal Science Nov. 2006NBCEC Brown Bagger
Former solution, probabilities
2
2
1
1
1
1
1
1
X
2
2
1
1
Prob (dwarf carrier/ relatives phenotype, genotype, individuals genotype)
1
1
1
1
Cow with known relatives withdwarf progeny and same haplotype
= dwarf carrier= normal
Department of Animal Science Nov. 2006NBCEC Brown Bagger
RN14HSA4 MMU5
MRPL1FRAS1ANXA3
BMP2KPAQR3
GK2GDEP
ANTXR2PRDM8FGF5
BMP3PRKG2
HNRPDMASASCD4THAP9
COPS4PLAC8 HPSEHEL308
Candidate genes from conserved syntenic blocks across human, mouse and rat contain
BTA
Department of Animal Science Nov. 2006NBCEC Brown Bagger
Selection of candidate genes
• Gene Function
• Knockout analysis in the mouse
• Naturally occurring mutants
• Based on these criteria, 4 genes were selected
Department of Animal Science Nov. 2006NBCEC Brown Bagger
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)
Department of Animal Science Nov. 2006NBCEC Brown Bagger
100 75 40 3330 98 (kB)3124714 1
1 2 3 4 5 6 7 8 9 10
Candidate genes genotyped
ID Position Alleles Frequency
AFR227 96.9cM 3 1=18%
2=57%
3=25%
BMP2K 5’ UTR A/G A=82%
G=18%
Intron 10 C/T C=44%
T=56%
3’ UTR C/T C=62%
T=38%
PRKG2 Intron 1 A/G A=40%
G=60%
Intron 2 C/T C=14%
T=86%
ID Position Alleles Frequency
PRKG2 Intron 3 A/G A=22%
G=78%
Intron 6 A/C A=38%
C=62%
Exon 15 C/T C=46%
T=54%
Intron 16 C/G C=42%
G=58%
Intron 17 A/T A=30%
T=70%
BM4311 97.7cM 3 1=24%
2=24%
3= 2%
Department of Animal Science Nov. 2006NBCEC Brown Bagger
Interval mapping
Marker Interval
Department of Animal Science Nov. 2006NBCEC Brown Bagger
A mutation in PRKG2 is likely causal
PRKG2=cGMP dependant, type II, protein kinase.
Wild-type/Unaffected
Carrier
Dwarf/affected
R PT R
R/Z PT R
ZT R
Department of Animal Science Nov. 2006NBCEC Brown Bagger
The PRKG2 mutation drastically changes protein structure
Protein kinase domain
762AA80 240 480
Α-helix domainNTP binding domain
Exon15 C/TPredicted PRKG2 in dwarves
678AAWild-typeMutant
A. B.
Department of Animal Science Nov. 2006NBCEC Brown Bagger
PRKG2 is necessary for long bone growth
Long Bone Length Growth Plate
Wildtype Knockout
Femur Transplants
Pfeifer et al. 1996
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Additional functional work
Male carrierFemale dwarf Male dwarf
Department of Animal Science Nov. 2006NBCEC Brown Bagger
Test for Angus dwarfism
50
55
60
65
70
75
80
85
Birth 1 Month
Dwarf
Carrier
Sh
ou
lder
Hei
gh
t in
In
ches
Department of Animal Science Nov. 2006NBCEC Brown Bagger
Collagen 2 (COL2) tranascript levels in Wild-type vs. Dwarf PRKG2 transfected HuH-7 cells
1.61.71.81.9
22.12.22.32.42.52.62.72.82.9
3
Control (pcDNA3) WTPRKG2 dwarf PRKG2Vector
Ln s
tarti
ng tr
ansc
ript l
evel
WT vs. control (P=0.39)
WT vs. dwarf (P=0.0028)
Dwarf vs. control (P=0.0028)N=4 reps/ trt
Department of Animal Science Nov. 2006NBCEC Brown Bagger
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
Department of Animal Science Nov. 2006NBCEC Brown Bagger
Acknowledgements
Iowa State James Koltes Bishnu Mishra University of NebraskaDr. Zhiliang Hu David Steffen
University of Liege University of SydneyMichel Georges Julie CavanaghWouter Coppieters Imke Tammen
Haruko Takeda Ohio State UniversityWashington State University Chuck Hines
Rowland Cobbold American Angus AssociationTawfik Aboellail Bryce Schumann