Related Experiment Video
Updated: Aug 14, 2025
Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis
Published on: June 9, 2018
Identification and characterization of two SERPINC1 mutations causing congenital antithrombin deficiency
Han-Lu Wang1, Dan-Dan Ruan1, Min Wu1
1Fujian Provincial Hospital, Shengli Clinical Medical College of Fujian Medical University, Fuzhou, 350001, China.
Insights
Two new SERPINC1 gene mutations, p.Asn460Thrfs*20 and p.Arg229*, cause hereditary antithrombin (AT) deficiency. These genetic variants lead to thrombotic disease through distinct molecular mechanisms, with p.Asn460Thrfs*20 being a novel discovery.
Area of Science:
- Genetics
- Molecular Biology
- Hematology
Background:
- Antithrombin (AT) is a key anticoagulant protein crucial for hemostasis.
- Hereditary AT deficiency, often caused by SERPINC1 mutations, is a rare genetic disorder predisposing individuals to venous thrombosis and pulmonary embolism.
Purpose of the Study:
- To investigate the clinical characteristics of two families with hereditary AT deficiency.
- To identify the causative genetic mutations in SERPINC1.
- To elucidate the functional consequences of identified mutations on AT protein structure and function.
Main Methods:
- Next-generation sequencing and Sanger sequencing were employed to screen for and confirm pathogenic variants in probands and their families.
- In vitro studies involved constructing and transfecting mutant and wild-type SERPINC1 plasmids into HEK293T cells to assess protein expression and localization.
- Bioinformatic analyses were performed to predict the structural and functional impact of the identified mutations.
Main Results:
- Two novel SERPINC1 variants, a frameshift mutation (c.1377delC; p.Asn460Thrfs*20) and a premature stop codon (p.Arg229*), were identified in two pedigrees with hereditary AT deficiency.
- Bioinformatic analysis suggested that p.Asn460Thrfs*20 disrupts protein structure by affecting disulfide bonds, while p.Arg229* leads to a truncated protein lacking critical functional domains.
- Cell-based assays demonstrated reduced AT protein expression for the c.1377delC mutation and a truncated protein for the c.685C > T (p.Arg229*) mutation, correlating with decreased AT activity and thrombophilia phenotypes.
Conclusions:
- The identified p.Asn460Thrfs*20 and p.Arg229* variants in SERPINC1 are causative for hereditary AT deficiency in the investigated pedigrees.
- These mutations impair antithrombin function through distinct molecular mechanisms.
- The p.Asn460Thrfs*20 variant represents a newly discovered cause of hereditary antithrombin deficiency.
Background:
Antithrombin (AT) is the main physiological anticoagulant involved in hemostasis. Hereditary AT deficiency is a rare autosomal dominant thrombotic disease mainly caused by mutations in SERPINC1, which was usually manifested as venous thrombosis and pulmonary embolism. In this study, we analyzed the clinical characteristics and screened for mutant genes in two pedigrees with hereditary AT deficiency, and the functional effects of the pathogenic mutations were evaluated.
Methods:
Candidate gene variants were analyzed by next-generation sequencing to screen pathogenic mutations in probands, followed by segregation analysis in families by Sanger sequencing. Mutant and wild-type plasmids were constructed and transfected into HEK293T cells to observe protein expression and cellular localization of SERPINC1. The structure and function of the mutations were analyzed by bioinformatic analyses.
Results:
The proband of pedigree A with AT deficiency carried a heterozygous frameshift mutation c.1377delC (p.Asn460Thrfs*20) in SERPINC1 (NM000488.3), a 1377C base deletion in exon 7 resulting in a backward shift of the open reading frame, with termination after translation of 20 residues, and a different residue sequence translated after the frameshift. Bioinformatics analysis suggests that the missing amino acid sequence caused by the frameshift mutation might disrupt the disulfide bond between Cys279 and Cys462 and affect the structural function of the protein. This newly discovered variant is not currently included in the ClinVar and HGMD databases. p.Arg229* resulted in a premature stop codon in exon 4, and bioinformatics analysis suggests that the truncated protein structure lost its domain of interaction with factor IX (Ala414 site) after the deletion of nonsense mutations. However, considering the AT truncation protein resulting from the p.Arg229* variant loss a great proportion of the molecule, we speculate the variant may affect two functional domains HBS and RCL and lack of the corresponding function. The thrombophilia and decreased-AT-activity phenotypes of the two pedigrees were separated from their genetic variants. After lentiviral plasmid transfection into HEK293T cells, the expression level of AT protein decreased in the constructed c.1377delC mutant cells compared to that in the wild-type, which was not only reduced in c.685C > T mutant cells but also showed a significant band at 35 kDa, suggesting a truncated protein. Immunofluorescence localization showed no significant differences in protein localization before and after the mutation.
Conclusions:
The p.Asn460Thrfs*20 and p.Arg229* variants of SERPINC1 were responsible for the two hereditary AT deficiency pedigrees, which led to AT deficiency by different mechanisms. The p.Asn460Thrfs*20 variant is reported for the first time.
