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Published on: September 9, 2012
Clinical and Molecular Characterization of Nine Novel Antithrombin Mutations
Judit Kállai1,2, Réka Gindele1, Krisztina Pénzes-Daku1
1Division of Clinical Laboratory Science, Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary.
Insights
This study identified nine novel antithrombin (AT) mutations, revealing six cause type I AT deficiency through impaired synthesis or secretion, and two suggest type II AT deficiency. The findings enhance understanding of AT deficiency's genetic basis.
Area of Science:
- Genetics and Molecular Biology
- Hematology and Thrombosis
Background:
- Antithrombin (AT) deficiency (ATD) is a severe inherited thrombophilia.
- Understanding novel SERPINC1 mutations is crucial for diagnosing and managing ATD.
- Genotype-phenotype correlations in ATD require further elucidation.
Purpose of the Study:
- To identify and characterize novel mutations in the SERPINC1 gene.
- To investigate the genotype-phenotype correlations of these new AT mutations.
- To elucidate the pathogenic mechanisms underlying different types of AT deficiency.
Main Methods:
- Expression of nine novel mutant antithrombin (AT) proteins in HEK293 cells.
- Detection of wild-type and mutant AT using Western blotting, N-Glycosidase F digestion, and ELISA.
- Analysis of AT mRNA expression via RT-qPCR, functional studies (AT activity, heparin binding via SPR), and in silico methods.
Main Results:
- Six novel mutations were confirmed to cause Type I ATD due to altered protein synthesis or secretion.
- Two mutations suggested Type II ATD, involving heparin-binding site defects or pleiotropic effects.
- The pathogenic role of one mutation (p.Arg14Lys) remained equivocal.
Conclusions:
- This study provides in vitro evidence for the pathogenic nature of novel SERPINC1 mutations.
- The findings contribute to a better understanding of the molecular basis of antithrombin deficiency.
- Characterization of these mutations aids in predicting clinical phenotypes and guiding patient management.
Abstract:
Antithrombin (AT) is the major plasma inhibitor of thrombin (FIIa) and activated factor X (FXa), and antithrombin deficiency (ATD) is one of the most severe thrombophilic disorders. In this study, we identified nine novel AT mutations and investigated their genotype-phenotype correlations. Clinical and laboratory data from patients were collected, and the nine mutant AT proteins (p.Arg14Lys, p.Cys32Tyr, p.Arg78Gly, p.Met121Arg, p.Leu245Pro, p.Leu270Argfs*14, p.Asn450Ile, p.Gly456delins_Ala_Thr and p.Pro461Thr) were expressed in HEK293 cells; then, Western blotting, N-Glycosidase F digestion, and ELISA were used to detect wild-type and mutant AT. RT-qPCR was performed to determine the expression of AT mRNA from the transfected cells. Functional studies (AT activity in the presence and in the absence of heparin and heparin-binding studies with the surface plasmon resonance method) were carried out. Mutations were also investigated by in silico methods. Type I ATD caused by altered protein synthesis (p.Cys32Tyr, p.Leu270Argfs*14, p.Asn450Ile) or secretion disorder (p.Met121Arg, p.Leu245Pro, p.Gly456delins_Ala_Thr) was proved in six mutants, while type II heparin-binding-site ATD (p.Arg78Gly) and pleiotropic-effect ATD (p.Pro461Thr) were suggested in two mutants. Finally, the pathogenic role of p.Arg14Lys was equivocal. We provided evidence to understand the pathogenic nature of novel SERPINC1 mutations through in vitro expression studies.
