A nonsense mutation in cGMP-dependent type II protein kinase (PRKG2) causes dwarfism in American Angus cattle

Abstract

Historically, dwarfism was the major genetic defect in U.S. beef cattle. Aggressive culling and sire testing were used to minimize its prevalence; however, neither of these practices can eliminate a recessive genetic defect. We assembled a 4-generation pedigree to identify the mutation underlying dwarfism in American Angus cattle. An adaptation of the Elston-Steward algorithm was used to overcome small pedigree size and missing genotypes. The dwarfism locus was fine-mapped to BTA6 between markers AFR227 and BM4311. Four candidate genes were sequenced, revealing a nonsense mutation in exon 15 of cGMP-dependant type II protein kinase (PRKG2). This C/T transition introduced a stop codon (R678X) that truncated 85 C-terminal amino acids, including a large portion of the kinase domain. Of the 75 mutations discovered in this region, only this mutation was 100% concordant with the recessive pattern of inheritance in affected and carrier individuals (log of odds score = 6.63). Previous research has shown that PRKG2 regulates SRY (sex-determining region Y) box 9 (SOX9)-mediated transcription of collagen 2 (COL2). We evaluated the ability of wild-type (WT) or R678X PRKG2 to regulate COL2 expression in cell culture. Real-time PCR results confirmed that COL2 is overexpressed in cells that overexpressed R678X PRKG2 as compared with WT PRKG2. Furthermore, COL2 and COL10 mRNA expression was increased in dwarf cattle compared with unaffected cattle. These experiments indicate that the R678X mutation is functional, resulting in a loss of PRKG2 regulation of COL2 and COL10 mRNA expression. Therefore, we present PRKG2 R678X as a causative mutation for dwarfism cattle.

Fig. 1.

The maximum LOD score for Angus dwarfism on BTA6 occurs at the C/T nonsense mutation, indicated by a black triangle, in exon 15 of PRKG2. Microsatellite markers are indicated by arrows; SNP markers are indicated by triangles.

Fig. 2.

Comparison of COL2 mRNA expression levels between WT btPRKG2 and R678X btPRKG2 transfected HUH-7 cells. The lines above the bar graph denote the accompanying statistical difference for the 6 treatment comparisons. The P value directly above each line is associated with the difference between only the outermost 2 bars underneath the line. All P values are Bonferroni corrected to account for multiple testing. NS, not significant.

Fig. 3.

In vivo gene expression of COL2 and COL10 was measured in bovine PRGK2^R678X/R678X (dwarf) and PRKG2^+/R678X (unaffected) tibia growth plates. (A) Difference in COL2 expression in dwarf R678X PRKG2 mutants versus normal cattle growth plate. (B) Difference in COL10 expression in dwarf R678X PRKG2 mutants versus normal cattle growth plate.