Submitted to: Veterinary Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/21/2005
Publication Date: 7/1/2006
Citation: Johnson, J.S., Laegreid, W.W., Basaraba, R.J., Baker, D.C. 2006. Truncated gamma-glutamyl carboxylase in Rambouillet sheep. Veterinary Pathology. 43(4):430-437.
Interpretive Summary: A severe bleeding disorder was observed in a flock of Rambouillet sheep. This resulted in the death of newborn lambs due to the trauma of birth. A vitamin-K dependent enzyme involved in maturation of clotting factors in blood was found to have reduced activity in affected lambs. This enzyme, gamma-glutamyl carboxylase, is the product of a gene designated GGCX. We sequenced GGCX in individuals from affected flocks, as well as the MARC Sheep Diversity Panel, and identified a single nucleotide polymorphism (SNP) that was strongly associated with the bleeding disorder. The SNP caused a shortened form of the enzyme to be produced with lowered activity. Tests for the SNP can now be used to identify carrier animals in flocks that exhibit this disorder.
Technical Abstract: A flock of Rambouillet sheep was examined because of lamb mortality due to ineffective hemostasis at parturition. Decreased activities of coagulation factors II, VII, IX, and X, severely reduced hepatic gamma-glutamyl carboxylase activity and adequate vitamin K 2,3 epoxide reductase activity was determined. Parenteral vitamin K1 supplementation did not improve vitamin K-dependent coagulation factor activities in three affected lambs. Affected lamb gamma-glutamyl carboxylase DNA was sequenced, and four single nucleotide polymorphisms (SNP’s 2-5) of the gamma-glutamyl carboxylase gene were identified. Single nucleotide polymorphism-4 results in an arginine to stop codon (UGA) substitution, which prematurely terminates the peptide at residue 686 (R686Stop). This genotype (GATT/GATT) has a strong association with the coagulopathy observed in clinically affected lambs, p<0.001. The frequency of SNP-3 in exon 11 (R486H) within the MARC 1.1 database is high in the U.S. sheep population overall. Gamma-glutamyl carboxylase activity in hepatic microsomes from a SNP-3 homozygous lamb lacking the SNP-4 mutation (GACC/GACC) was similar to control sheep homozygous for arginine at 486 and also lacking SNP-4 (TGCC/TGCC) indicating the R486H does not measurably impact gamma-glutamyl carboxylase activity. The remaining two SNPs (2 and 5) are located within non-coding intron sequences. These four SNPs allowed for determining the genotype associated with the observed fatal coagulopathy. Screening for the premature truncation (SNP-4) based on the presence of Bbv I restriction site in clinically normal lambs, but not in the homozygous affected lambs allows for detection of the heterozygous state (GATT/GACC), as carrier animals are clinically normal.