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Truncated Gamma-Glutamyl Carboxylase in Rambouillet Sheep

Colorado State University, College of Veterinary Medicine and Biomedical Sciences, Department of Microbiology, Immunology, and Pathology, Fort Collins, CO (JSJ, RJB, DCB), US Department of Agriculture, Agricultural Research Service, US Meat Animal Research Center, Clay Center, NB (WSL)

A flock of Rambouillet sheep was examined because of increased lamb mortality due to ineffective hemostasis at parturition. Decreased activities of coagulation factors II, VII, IX, and X, and severely reduced hepatic -glutamyl carboxylase activity with adequate vitamin K 2,3 epoxide reductase activity was determined.^1,21 Parenteral vitamin K₁ supplementation did not improve vitamin K-dependent coagulation factor activities in 3 affected lambs. Affected lamb -glutamyl carboxylase deoxyribonucleic acid was sequenced, and 4 single nucleotide polymorphisms (SNPs 2–5) of the -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 US 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 that the R486H does not measurably impact -glutamyl carboxylase activity. The remaining two SNPs (2 and 5) are located within non-coding intron sequences. These 4 SNPs allowed for determining the genotype associated with the observed fatal coagulopathy. Screening for the premature truncation (SNP-4) based on the presence of a Bbv I restriction site in clinically normal lambs but not in the homozygous affected lambs allows for detection of the heterozygous state (GATT/GACC), because carrier animals are clinically normal.

Key words: Gamma-glutamyl carboxylase; hemostasis; sheep; vitamin K.

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