Identification and characterization of two SERPINC1 mutations causing congenital antithrombin deficiency

Abstract

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.

In vivo LNP-CRISPR Approaches for the Treatment of Hemophilia

“…1). The inhibition of anticoagulant proteins increases thrombin generation, which can induce thrombosis [47][48][49][50]. Based on this mechanism, rebalancing therapy is an alternative to replacing deficient coagulation factors by suppressing the expression and function of anticoagulants in the blood to restore the blood coagulation ability [51].…”

Antithrombin Therapy: Current State and Future Outlook

“…The advent of next-generation sequencing has led to an explosion in investigations of SERPINC1 variants of both type I and type II, most often initiated to explore cases of familial thrombosis. 31,39–41…”

“…After sequencing, bioinformatic analyses are used to link variant genotypes to the structure and function of the AT protein and to detect associations between protein defects and disease phenotypes. 39–41 Recent work points to the importance of environmental factors on the phenotype of some variants, with a new potential classification termed transient hereditary AT deficiency. 42 These genotypic/phenotypic studies and molecular analyses may eventually have potential in the prognostication of VTE, but their interpretation is currently nuanced and direct clinical applications are not clear.…”