PB0622 - Determining the dominant-negative causality of FXIII heterozygous mutations using a bi-cistronic, bi-tagged expression system

Background: Mild Factor XIII (FXIII) deficiency (FXIII activity 20-60%) is a phenomenon resulting from heterozygous mutations in either F13A1 or F13B genes coding for the plasma circulating coagulation FXIII. Mild FXIII deficiency is asymptomatic unless the individual is exposed to physical trauma i.e. peri-operatively, dental extraction, etc. Earlier, we reported 23 missense mutations from mild FXIII deficient individuals. However, the “dominant-negative” effect of these mutations in a heterozygous scenario can only be proved by studying the heterozygous variant species in isolation.

Aims: Our study aims to use a bi-cistronic bi-tagged expression system in which both mutant and wild-type alleles of all 23 missense mutations can be simultaneously expressed followed by the isolation of heterozygous species for further analysis.

Methods: Two types of expression vector (Figure 1) were developed for this analysis: 1) A two promoter system with a CMV promoter separated by SV40 PA translation-stop site in which the wildtype and the mutant are cloned after each promoter and 2) P2A vector system in which the cloned mutant and wild type are separated P2A self-cleaving peptide. Both the mutant and wild type ORFs were tagged with His and Strep tags at the N and C terminal respectively to allow us to systematically segregate wild type, heterozygous, and mutant homozygous species post transient transfection onto HEK293t cells followed by upscaling in HEK293F suspension culture.

Results: Almost all mutations showed close to 50-80% of FXIII activity as well as antigenic levels corresponding to that of the wild type in concordance with the FXIII levels observed in the mild FXIII deficient individuals carrying these mutations. Further purification and functional results are awaited.

Conclusion(s): Both systems were able to successfully express the wild type as well as the mutant alleles as indicated by the carrier/ heterozygous activity levels of the mutant.