A spontaneous hereditary coagulopathy in Rambouillet sheep

Abstract

An inbred flock of Rambouillet sheep was investigated because of increased lamb mortality due to ineffective periparturient hemostasis. Affected, term, neonatal lambs had extensive subcutaneous, umbilical, and intra-cavitary hemorrhage that resulted in hypovolemia and death. Affected lambs were identified by prolongation of the activated clotting time, one-stage prothrombin time (PT), and activated partial thromboplastin time (aPTT). Decreased activity of vitamin K-dependent coagulation factors II, VII, IX, and X was present in all affected lambs while non-vitamin K-dependent coagulation factor activities were similar between affected and unaffected lambs. The hemorrhagic diathesis could not be corrected by supraphysiologic administration of vitamin K1; however, vitamin K, did prolong the required transfusion interval for maintaining adequate hemostasis. Defective γ-glutamyl carboxylase was considered the most likely cause of the heritable coagulopathy because of the inability to correct the hemorrhagic state with supraphysiologic vitamin K1, and the lack of reported skeletal abnormalities observed in human cases of epoxide reductase deficiency or in utero warfarin exposure. To confirm the suspected underlying cause of abnormal coagulation, crude liver microsomes were isolated from affected lamb liver and evaluated for hepatic γ-glutamyl carboxylase and vitamin K-epoxide reductase activities. Hepatic γ-glutamyl carboxylase activity was significantly decreased compared to age and sex matched control lambs while vitamin K 2,3 epoxide reductase activity was similar between affected and control lamb liver microsomes. We sequenced cDNA and genomic DNA from affected lambs, carrier sheep, and control lambs, and identified four single nucleotide polymorphisms (SNPs) of the γ-glutamyl carboxylase gene allowing for haplotype and genotype characterization. Two of these SNPs were located within the coding sequence of the γ-glutamyl carboxylase gene, specifically in exon 10 (R486H) and exon 14 (R686Stop). Enzyme kinetics demonstrates that neither mutation R486H nor R686Stop significantly impacts the interaction of the enzyme for the synthetic substrate Phe-Leu-Glu-Glu-Leu (FLEEL), nor vitamin K-dependent propeptides of coagulation factor II, IX, and X. Therefore these reactions occur nearer the NH2- terminus than residue 686. Through comparisons of these SNP frequencies within our inbred sheep flock and the larger U.S. sheep population we determined the likely causative mutation resulting in the fatal coagulopathy to be the result of a premature truncation of the γ-glutamyl carboxylase protein (R686Stop), p<0.001. This sheep flock represents the only viable animal model of hereditarily defective γ-glutamyl carboxylase. Two human case reports of γ-glutamyl carboxylase deficiency and site directed mutagenesis have laid the foundation for determining critical amino acids necessary for enzymatic function, and propagation of this animal model will increase our understanding of the impact of vitamin K metabolism on various systems in the body.