Characterisation of two naturally occuring coagulation factor VII variants (ALA244V AL AND PR0134THR)

Abstract

Pre-operative coagulation studies on a 4-year old girl indicated the presence of coagulation factor VII deficiency in a kindred from Malta. Factor VII assays with a panel of thromboplastins from four different species, and factor VII antigen assays, indicated phenotypic heterogeneity within the kindred. Sequence analysis of the factor VII coding region and 5' untranslated region demonstrated the presence of two new missense mutations. One of these was a cytosine to adenine transversion at position 8,906. This mutation, designated factor VII Malta I, leads to the substitution of the proline at position 134 by threonine. The other mutation was a cytosine to thymine transition at position 10,648. This mutation, designated factor VII Malta II, leads to the substitution of the alanine at position 244 by valine. Sequence analysis also showed that the factor VII alle1es of the kindred were of two different frameworks, framework 1 and framework 2. Framework 2 is known to be associated with decreased levels of factor VII coagulant activity, factor VII antigen and activated factor VII. The factor VII Malta I mutation occurred on a framework 1 allele, whereas the factor VII Malta II mutation occurred on a framework 2 allele. In all, four factor VII alle1es

segregated in the kindred. These were: (i) a wild-type framework 1 allele, (ii) a wild- type framework 2 allele, (iii) a framework I-factor VII Malta I allele, and (iv) a

framework 2-factor VII Malta II allele. These four alle1es resulted in a complex pattern of genotypic combinations, which accounted for the phenotypic heterogeneity among members of the kindred. Both the factor VII Malta I and the factor VII Malta II mutations were likely associated with a cross-reacting material reduced defect.

In order to investigate the effect of the two amino acid substitutions on the framework 1 gene product, framework 1 factor VII cDNAs coding for factor VII Malta I and factor VII Malta II were constructed and expressed in Chinese hamster ovary cells. The specific activity of the factor VII Malta I recombinant variant did not differ significantly from that of recombinant wild-type factor VII expressed by the wild-type framework 1 factor VII cDNA. On the other hand, the factor VII Malta II recombinant variant had a specific activity slightly decreased as compared to that of the recombinant wild-type protein. Both variants were fully activated by activated factor X. Furthermore, the Kcat and KM for activation of factor X in the presence of tissue factor, by activated recombinant factor VII Malta I and by activated recombinant factor VII Malta II, did not differ significantly from those by activated wild-type factor.

The data obtained excluded gross dysfunctional effects of the two ammo acid substitutions on the functional characteristics of the framework 1 gene product. However, the studies done to date suggest a complex defect perhaps due to a small decrease in activity, defective secretion, decreased stability, or sequestration in an inactive complex in plasma.