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Discovery of a putative causal
mutation for Angus dwarfism
Department of Animal Science
NBCEC Brown Bagger
Nov. 2006
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
NBCEC Brown Bagger
Nov. 2006
Calves at 7 months vs. birth
Two bulls at Birth
7 Months
16cM
Dwarf
C/T
T/T
Normal
Dwarf
male
Department of Animal Science
18cM
Normal
T/T
Dwarf
female
NBCEC Brown Bagger
Nov. 2006
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
NBCEC Brown Bagger
Nov. 2006
Family Structure
Sire 1
Sire 2
Dam 6
Sire 5
Sire 10
Dam 11
Dwarf1
Department of Animal Science
Dam 12
Dwarf2
Sire 3
Dam 7
Dam 13
Dwarf3
Dam 4
Dam 8
Dam 14
Dwarf4/5
Dam 9
Sire 15
Sire 16
Dwarf6
NBCEC Brown Bagger
Nov. 2006
Pedigree Analysis
Department of Animal Science
NBCEC Brown Bagger
Nov. 2006
Are known bovine mutations
responsible Angus dwarfism?
1356
TTTTCAAAGG C GAGCTGAAGC - Normal limbin
TTTTCAAAGG T GAGCTGAAGC - Mutant limbin
TTTTCAAAGG C GAGCTGAAGC - Angus dwarf/normal
2054/2055
AGGGACCTGG CA GCCAGGGCCG - Normal limbin
AGGGACCTGG G GCCAGGGCCG - Mutant limbin
AGGGACCTGG CA GCCAGGGCCG – Angus dwarf/normal
Mishra et al., 2003
Department of Animal Science
NBCEC Brown Bagger
Nov. 2006
Are known bovine mutations
responsible Angus dwarfism?
Department of Animal Science
NBCEC Brown Bagger
Nov. 2006
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
NBCEC Brown Bagger
Nov. 2006
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
NBCEC Brown Bagger
Nov. 2006
Initial Results of Linkage
mapping
Department of Animal Science
NBCEC Brown Bagger
Nov. 2006
Improved Marker Map Coverage
BTA 6
BTA 6
Marker 1
40 cM
Interval
8
Known dwarf
Gene = Limbin
Department of Animal Science
NBCEC Brown Bagger
Nov. 2006
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
NBCEC Brown Bagger
Nov. 2006
Former solution, probabilities
Cow with known
relatives with
dwarf progeny
and same
haplotype
= dwarf carrier
= normal
X
1
1
2
2
1
1
1
1
Prob (dwarf carrier/ relatives
phenotype, genotype, individuals
genotype)
1
1
2
2
Department of Animal Science
1
1
1
1
NBCEC Brown Bagger
Nov. 2006
Candidate genes from conserved
syntenic blocks across human,
mouse and rat contain
BTA
HSA4
THAP9
SCD4
MMU5
RN14
PLAC8
HEL308
HPSE
COPS4
MASA
HNRPD
PRKG2
BMP3
ANTXR2
FGF5
PRDM8
GDEP
GK2
PAQR3
Department of Animal Science
BMP2K
ANXA3
FRAS1
MRPL1
NBCEC Brown Bagger
Nov. 2006
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
NBCEC Brown Bagger
Nov. 2006
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
NBCEC Brown Bagger
Nov. 2006
Candidate genes genotyped
1
2
100
75
40
3
4
5
6
7
330
3
1
14
8
47
9
12 3
10
98 (kB)
ID
Position
Alleles
Frequency
ID
Position
Alleles
Frequency
AFR227
96.9cM
3
1=18%
2=57%
3=25%
PRKG2
Intron 3
A/G
A=22%
G=78%
Intron 6
A/C
A=82%
G=18%
A=38%
C=62%
Exon 15
C/T
C=44%
T=56%
C=46%
T=54%
Intron 16
C/G
C=62%
T=38%
C=42%
G=58%
Intron 17
A/T
A=30%
T=70%
97.7cM
3
1=24%
2=24%
3= 2%
BMP2K
5’ UTR
Intron 10
3’ UTR
PRKG2
Intron 1
Intron 2
A/G
C/T
C/T
A/G
C/T
Department of Animal Science
A=40%
G=60%
C=14%
T=86%
BM4311
NBCEC Brown Bagger
Nov. 2006
Interval mapping
Marker Interval
Department of Animal Science
NBCEC Brown Bagger
Nov. 2006
A mutation in PRKG2 is likely
causal
PRKG2=cGMP dependant, type II, protein kinase.
Wild-type/
Unaffected
T
R
R
P
T
R
R/Z
P
T
R
Z
Carrier
Dwarf/
affected
Department of Animal Science
NBCEC Brown Bagger
Nov. 2006
The PRKG2 mutation drastically
changes protein structure
B.
A.
Α-helix domain
80
NTP binding domain Protein kinase domain
240
480
762AA
Predicted PRKG2 in dwarves
678AA
Department of Animal Science
Exon15
C/T
Mutant
Wild-type
NBCEC Brown Bagger
Nov. 2006
PRKG2 is necessary for long bone
growth
Long Bone Length
Femur Transplants
Growth Plate
Wildtype
Knockout
Pfeifer et al. 1996
Department of Animal Science
NBCEC Brown Bagger
Nov. 2006
Additional functional work
Female dwarf
Department of Animal Science
Male dwarf
Male carrier
NBCEC Brown Bagger
Nov. 2006
Shoulder Height in Inches
Test for Angus dwarfism
85
80
Dwarf
Carrier
75
70
65
60
55
50
Birth
Department of Animal Science
1 Month
NBCEC Brown Bagger
Nov. 2006
Ln starting transcript level
3
2.9
2.8
2.7
2.6
2.5
2.4
2.3
2.2
2.1
2
1.9
1.8
1.7
1.6
Collagen 2 (COL2) tranascript levels in Wild-type vs. Dwarf
PRKG2 transfected HuH-7 cells
N=4 reps/ trt
Dwarf vs. control (P=0.0028)
WT vs. dwarf (P=0.0028)
WT vs. control (P=0.39)
Control (pcDNA3)
Department of Animal Science
WTPRKG2
Vector
dwarf PRKG2
NBCEC Brown Bagger
Nov. 2006
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
NBCEC Brown Bagger
Nov. 2006
Acknowledgements
Iowa State
James Koltes Bishnu Mishra
Dr. Zhiliang Hu
University of Nebraska
University of Liege
University of Sydney
Michel Georges
Wouter Coppieters
Haruko Takeda
Julie Cavanagh
Imke Tammen
David Steffen
Ohio State University
Washington State University Chuck Hines
Rowland Cobbold
American Angus Association
Tawfik Aboellail
Department of Animal Science
Bryce Schumann
NBCEC Brown Bagger
Nov. 2006