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To cite this article: Callan MB, Aljamali MN, Margaritis P, Griot-Wenk ME, Pollak ES, Werner P, Giger U, High KA. A novel missense mutation responsible for factor VII deficiency in research Beagle colonies. J Thromb Haemost 2006; 4: 2616-22.Summary. Background: Canine factor VII (cFVII) deficiency, an autosomal recessive trait originally identified in research Beagles, is associated with a mild to moderate bleeding tendency. Objective: Our aim was to identify and characterize the mutation causing cFVII deficiency. Methods: In order to sequence the coding regions of the cFVII gene, we cloned the cFVII cDNA. Genomic DNA and plasma from FVIIdeficient Beagles and obligate carriers were utilized. Results: In all FVII-deficient dogs, we identified a single causative G to A missense mutation in exon 5, encoding the second epidermal growth factor-like domain, resulting in substitution of glycine 96 by glutamic acid, with plasma FVII coagulant activity of £ 4% in affected Beagles. In vitro expression indicated that the majority (96%) of cFVII-G96E protein was retained intracellularly. In addition, analysis of purified recombinant wild-type and mutant cFVII proteins demonstrated reduced activity of the mutant (< 2%) compared with wild-type. Rotational thromboelastometry revealed a severe impairment of clotting activity in affected Beagles, and heterozygotes also exhibited changes in coagulation-based assays. Using a mutation-specific polymerase chain reaction/ restriction digest that allows rapid identification of the G96E mutation, we surveyed a US research Beagle colony and identified a mutant allelic frequency of 31%. Conclusions: We have identified a single causative mutation for cFVII deficiency that may have implications for pharmacotoxicologic research, because reduced FVII coagulant activity may alter hemostatic and/or cardiovascular endpoints in this commonly used animal species.

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SummaryThe biochemical nature of the feline AB blood group system was characterized by analysing red blood cells from homozygous (genotype A/A) and heterozygous (A/B) type A, type B (B/B), and type AB cats. High performance thin layer chromatography (HPTLC) of red cell gly‐colipids revealed that specific neuraminic acids (NA) on gangliosides, containing ceramide dihexoside (CDH) as a backbone, correlated with the feline AB blood group antigens. Although disialogangliosides predominated, mono‐ and trisialogangliosides were also isolated. B cats expressed solely N‐acetyl‐NA (NeuNAc) on these gangliosides. In addition to expressing N‐glycolyl‐NA (NeuNGc) containing gangliosides, A red cells have gangliosides with only NeuNAc or mixtures of both NA. HPTLC profiles of disialogangliosides from homozygous and heterozygous A cats differed slightly in the quantity of disialogangliosides. Equal amounts of NeuNAc and NeuNGc containing disialogangliosides, as well as two intermediary forms, were recovered from AB erythrocytes. Analysing disialogangliosides from red cells belonging to 17 genetically related cats, we consistently obtained the expected disialoganglio‐side profile, based on blood typing and pedigree information. SDS‐PAGE of red cell membrane proteins and blotting with Triticum vulgaris, a lectin recognizing NeuNAc, revealed glycoproteins of approximately 51, 53, and 80 kD in B and AB cats but only a faint band of approximately 53 kD in A cats. By haemagglutination, Triticum vulgaris could also distinguish different blood types by specifically binding to B and AB cells. Flow cytometry showed that more anti‐B bound to B cells than anti‐A bound to A cells. Although AB cells had a broad range of fluorescence when compared to the profiles seen with A and B cells, the mean fluorescence with AB cells was half of that seen with A or B. These results further characterize the antigens determining the feline AB blood group system illustrating differences between A, B and AB cats, and between homozygous and heterozygous A cats.