Discovery of a putative causal mutation for Angus dwarfism

Department of Animal Science Nov. 2006NBCEC Brown Bagger

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

• Abnormal osteocyte phenotype


Calves at 7 months vs. birth

16cM 18cM

T/TT/TC/T

Normal Dwarffemale

Dwarfmale

7 Months

Two bulls at Birth

NormalDwarf


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


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


Pedigree Analysis


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


Are known bovine mutations responsible Angus dwarfism?


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


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


Initial Results of Linkage mapping


Improved Marker Map Coverage

Marker 1

BTA 6

BTA 6

40 cM

Known dwarf Gene = Limbin

Interval 8


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


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


RN14HSA4 MMU5

MRPL1FRAS1ANXA3

BMP2KPAQR3

GK2GDEP

ANTXR2PRDM8FGF5

BMP3PRKG2

HNRPDMASASCD4THAP9

COPS4PLAC8 HPSEHEL308

Candidate genes from conserved syntenic blocks across human, mouse and rat contain

BTA


Selection of candidate genes

• Gene Function

• Knockout analysis in the mouse

• Naturally occurring mutants

• Based on these criteria, 4 genes were selected


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)


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%


Interval mapping

Marker Interval


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


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.


PRKG2 is necessary for long bone growth

Long Bone Length Growth Plate

Wildtype Knockout

Femur Transplants

Pfeifer et al. 1996


Additional functional work

Male carrierFemale dwarf Male dwarf


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


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


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


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

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Discovery of a putative causal mutation for Angus dwarfism

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