Determining the causality of heterozygous missense mutations detected in F13A1 and F13B genes and reported from patients with mild coagulation factor XIII (FXIII) deficiency

A. Biswas1, A. Thomas1, J. Dodt2, H. Philippou3, E. Heathershaw3, H. E. Jürgen1, V. Ivaskevicius1, J. Oldenburg1 (1Bonn, Germany, 2Langen, Germany, 3Leeds, UK)

Bleeding disorders, coagulation and fibrinolytic factors
Date: 17.02.2017,
Time: 17:15 - 18:15

Objective: To evaluate the causality of heterozygous missense mutations reported in F13A1 and F13B genes reported from patients with mild coagulation Factor XIII (FXIII) deficiency by expressing them in heterologous cell lines.

Methods: The previously reported 16 F13A1 and 7 F13B missense mutations were cloned in mammalian expression vectors carrying F13A1 and F13B cDNAs and transfected into Cos-1 and HEK293T cell lines respectively. Transfections were also performed in combination with the wild type in order to mimic the heterozygous genotype. The intracellular-lysates/extracellular medium were collected after a transient transfection period and tested with a variety of assays. The B subunit mutations were tested for their secretion pattern (by confocal microscopy) and their ability to bind to A subunit. The A subunit mutations were tested by a variety of activity assays i.e. photometric activity assays, physiological incorporation assay, α-2 antiplasmin incorporation assay, non proteolytic activation(with only high concentration of calcium and no thrombin), fibrin crosslinking and clot thickness using scanning electron microscopy. Structural modeling/docking combined with simulation based analysis were performed on available crystals structures of FXIIIA subunit and homology based models of FXIIIB subunit sushi domains to determine the putative structural impact of the reported mutations.

Results: Apart from one FXIIIB subunit mutation i.e. p.Cys5Arg which showed a true secretion related defect, the remaining mutations showed either antigenic instability or lack of interaction with FXIIIA subunit. The effect of FXIIIA subunit mutations could be categorized into mild moderate and severe forms based on their in vitro expression phenotype. Two substitutions at the thrombin cleavage site showed type II FXIIIA deficiency i.e. they showed reduced activities associated with moderate or no change in antigen levels. Majority of the FXIIIA missense mutations showed varied effects on alpha-2-antiplasmin incorporation, clot thickness and fibrinogen cross-linking.

Conclusion: Our analysis demonstrates that these mutations influence separate structural and functional aspects of the coagulation FXIII.