Characterization of a novel SERPINC1 gene mutation (c.1247dupC) resulting in a secretion defect and type I antithrombin deficiency

Antithrombin (AT), encoded by the SERPINC1 gene, acts as an important anticoagulant factor in hemostasis by inactivating coagulation factors IIa (thrombin), IXa, Xa, XIa and XIIa. AT deficiency poses a major risk for thromboembolic events. We identified a novel SERPINC1 frameshift mutation (c.1247dupC) in two sisters with confirmed AT deficiency and clinically relevant thrombosis and examined the effect of this mutation on cellular and molecular level [Ruf, M. et al. (2024): SERPINC1 c.1247dupC: a novel SERPINC1 gene mutation associated with familial thrombosis results in a secretion defect and quantitative antithrombin deficiency. Thrombosis J. 22, 19. https://doi.org/10.1186/s12959-024-00589-5].

The SERPINC1 mutation c.1247dupC, resulting in a frameshift in SERPINC1 coding sequence and subsequently altering the amino acid sequence of the AT C-Terminus (p.Ser417LysfsTer48), was identified by Next Generation Sequencing.

Plasmids for the expression of wild-type and mutated SERPINC1 coding sequence fused to green fluorescent protein (GFP) or hemagglutinin tag (HA) were generated for transfection of HEK293T cells. Subcellular localization and secretion of the respective fusion proteins were analyzed by confocal laser scanning microscopy and Western blot.

The altered C-terminal sequence, caused by frameshift mutation p.Ser417LysfsTer48, affects the secretion of the mutated AT antigen. Since the GFP fusions of both wild-type and mutated AT are localized in the endoplasmic reticulum (ER) of the respective HEK293T cell lines, wild-type as well as mutated AT are able to enter the secretory pathway. In contrast to that, Western Blot analysis comparing cell lysates and cell culture supernatants of HEK293T cells transfected with constructs for GFP- or HA-tagged wild-type and mutated AT showed a strongly reduced secretion of the AT antigen with the p.Ser417LysfsTer48 mutation, indicating that the mutated AT is not able to pass the quality control of the ER.

The p.Ser417LysfsTer48 mutation results in impaired secretion, which might be caused by polymerization induced by aberrant folding or by new cysteine residues introduced by the frameshift mutation, providing free thiol groups that are recognized by the thiol-dependent ER quality control. The here described mutation is causative for the type I AT deficiency observed in the two patients.

There are no conflicts of interest.