Novel Mutation p.Asp374Val of SERPINC1 in a Turkish Family with Inherited Antithrombin Deficiency
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ABSTRACT:
SUBMITTER: Aslan D
PROVIDER: S-EPMC8171213 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundAntithrombin (AT) is an important anticoagulant in hemostasis. We describe here the characterization of a novel AT mutation associated with clinically relevant thrombosis. A pair of sisters with confirmed type I AT protein deficiency was genetically analyzed on suspicion of an inherited SERPINC1 mutation. A frameshift mutation, c.1247dupC, was identified and the effect of this mutation was examined on the cellular and molecular level.MethodsPlasmids for the expression of wild-type (WT) and mutated SERPINC1 coding sequence (CDS) fused to green fluorescent protein (GFP) or hemagglutinin (HA) tag were transfected into HEK293T cells. Subcellular localization and secretion of the respective fusion proteins were analyzed by confocal laser scanning microscopy and Western blot.ResultsThe c.1247dupC mutation results in a frameshift in the CDS of the SERPINC1 gene and a subsequently altered amino acid sequence (p.Ser417LysfsTer48). This alteration affects the C-terminus of the AT antigen and results in impaired secretion as confirmed by GFP- and HA-tagged mutant AT analyzed in HEK293T cells.ConclusionThe p.Ser417LysfsTer48 mutation leads to impaired secretion, thus resulting in a quantitative AT deficiency. This is in line with the type I AT deficiency observed in the patients.
| S-EPMC10860291 | biostudies-literature
Project description:Antithrombin (AT) deficiency is a blood disorder associated with an increased tendency to form thrombosis. Hereditary AT deficiency is frequently caused by mutations in SERPINC1 gene. It is usually inherited as an autosomal dominant with variable penetrance. Homozygous pathogenic mutations in this gene are extremely rare. We present a case of a 7-year-old female who presented at age of 4 years with massive cerebral sinus venous thrombosis. Thrombophilia workup showed a low AT level of 30%. Targeted genetic sequencing of SERPINC1 revealed a novel pathogenic homozygous mutation c.1320C>G p. (Phe440Leu). The patient was managed initially with unfractionated heparin with AT replacement using fresh frozen plasma and was later switched to only low-molecular-weight heparin. There was no recurrence or new thrombosis with 3 years of follow-up.
| S-EPMC10139800 | biostudies-literature
Project description:BackgroundAntithrombin (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.MethodsCandidate 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.ResultsThe 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.ConclusionsThe 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.
| S-EPMC9830717 | biostudies-literature
Project description:BackgroundInherited AT deficiency is an autosomal-dominant thrombophilic disorder usually caused by various SERPINC1 defects associated with a high risk of recurrent venous thromboembolism. In this article, the phenotype, gene mutation, and molecular pathogenic mechanisms were determined in three pedigrees with inherited AT deficiency.MethodsCoagulation indices were examined on STAGO STA-R-MAX analyzer. The AT:Ag was analyzed by ELISA. All exons and flanking sequences of SERPINC1 were amplified by PCR. AT wild type and three mutant expression plasmids were constructed and then transfected into HEK293FT cells. The expression level of AT protein was analyzed by ELISA and Western blot.ResultsThe AT:A and AT:Ag of probands 1 and 3 were decreased to 49% and 52 mg/dL, 38% and 44 mg/dL, respectively. The AT:A of proband 2 was decreased to 32%. The SERPINC1 gene analysis indicated that there was a p.Ile421Thr in proband 1, a p.Leu417Gln in proband 2, and a p.Met252Thr in proband 3, respectively. The AT mRNA expression level of the three mutants was not significantly different from AT-WT by qRT-PCR. The results of ELISA and Western blot tests showed that the AT-M252T and AT-I421T mutants had a higher AT expression than the AT wild type (AT-WT), and the AT protein expression of AT-L417Q mutants had no significant difference compared with AT-WT in the cell lysate. The AT expression levels of AT-M252T and AT-I421T mutants were lower than that of AT-WT, and there was no significant difference between AT-L417Q mutant and AT-WT in the supernatant.ConclusionThe p.I421T and p.M252T mutations affected the secretion of AT protein leading to type I AT deficiency of probands 1 and 3. The p.Leu417Gln mutation was responsible for the impaired or ineffective activity AT protein in proband 2 and caused type II AT deficiency.
| S-EPMC9701880 | biostudies-literature
Project description:ObjectiveAntithrombin (AT) plays a critical role in the coagulation system, and its deficiency induces hypercoagulability. AT deficiency is caused not only by inherited variants in the SERPINC1 gene but also by acquired conditions. Therefore, AT deficiency alone could not ensure the presence of the SERPINC1 mutation. We evaluated the utility of the SERPINC1 gene test in ischemic stroke, an important clinical type of arterial thrombosis.MethodsThis retrospective, observational study investigated symptomatic patients who underwent the SERPINC1 gene test because of decreased AT activity (<80%) during 2009-2021 at a tertiary hospital. For the detection of sequence variants in the SERPINC1 gene, direct Sanger sequencing and multiplex ligation-dependent probe amplification were performed. The phenotypes of patients with SERPINC1 gene mutations were examined, and the conditions associated with the pathogenic variants were analyzed.ResultsIn our cohort (n = 19), 13 of 19 patients (68.4%) had the pathogenic variant of the SERPINC1 gene. Ischemic stroke (n = 7) was significantly associated with the pathogenic variants (p = 0.044), and the pathogenicity detection rate was 100%. For any kind of arterial thrombosis (n = 8), the detection rate of the pathogenic variant was 87.5%, but was not statistically significant (p = 0.177). The detection rates of the pathogenic variant in ischemic stroke or arterial thrombosis groups were both higher than those in the venous thrombosis-only group (54.5%).ConclusionThe SERPINC1 gene test was useful in determining the cause of AT deficiency-related arterial thrombosis, especially ischemic stroke. We propose the diagnostic flow of SERPINC1-related ischemic stroke.
| S-EPMC9203840 | biostudies-literature
Project description:Antithrombin is a crucial anticoagulant serpin whose even moderate deficiency significantly increases the risk of thrombosis. Most cases with antithrombin deficiency carried genetic defects affecting exons or flanking regions of SERPINC1.We aimed to identify regulatory mutations inSERPINC1 through sequencing the promoter, intron 1 and 2 of this gene in 23 patients with antithrombin deficiency but without known genetic defects. Three cases with moderate antithrombin deficiency (63-78%) carried potential regulatory mutations. One located 200 bp before the initiation ATG and two in intron 1. These mutations disrupted two out of five potential vitamin D receptor elements (VDRE) identified in SERPINC1 with different software. One genetic defect, c.42-1060_-1057dupTTGA, was a new low prevalent polymorphism (MAF: 0.01) with functional consequences on plasma antithrombin levels. The relevance of the vitamin D pathway on the regulation of SERPINC1 was confirmed in a cell model. Incubation of HepG2 with paricalcitol, a vitamin D analog, increased dose-dependently the levels of SERPINC1transcripts and antithrombin released to the conditioned medium. This study shows further evidence of the transcriptional regulation of SERPINC1 by vitamin D and first describes the functional and pathological relevance of mutations affecting VDRE of this gene. Our study opens new perspectives in the search of new genetic defects involved in antithrombin deficiency and the risk of thrombosis as well as in the design of new antithrombotic treatments.
| S-EPMC4803246 | biostudies-literature
Project description:Antithrombin (AT) deficiency is an autosomal dominant disorder, and identification of mutation AT variants would improve our understanding of the anticoagulant function of this serine protease inhibitor (SERPIN) and the molecular pathways underlying this disorder. In the present study, we performed whole-exome sequencing of a Chinese family with deep vein thrombosis, and identified a new small deletion that eliminates four amino acids (INEL) from exon 4 of SERPINC1 gene. This causes type I AT deficiency by enhancing the intracellular retention of this protein. AT retention leads to endoplasmic reticulum (ER) stress, which further inhibits AT release. In addition, ER stress activates ER-associated degradation, which promotes AT degradation. Suppression of ER stress enhanced the secretion of AT, while inhibition of ER-associated degradation suppressed AT release. Thus, our study identified a new mutation (INEL deletion) causing type I AT deficiency, and uncovered a novel mechanism for AT retention through enhanced ER stress, which may provide an innovative approach for treating AT deficiency.
| S-EPMC5363556 | biostudies-literature
Project description:Despite the essential anticoagulant function of antithrombin and the high risk of thrombosis associated with its deficiency, the prevalence of antithrombin deficiency among patients with venous thromboembolism (VTE) is very low. However, increasing evidence suggests that antithrombin deficiency may be underestimated. The analysis of SERPINC1, the gene encoding antithrombin, in 1,304 consecutive Chinese VTE patients and 1,334 healthy controls revealed a hotspot involving residues 294 and 295 that severely increases the risk of VTE. We detected the c.883G>A (p.Val295Met) (rs201381904) mutation in 11 patients and just one control (OR = 13.6; 95% CI: 1.7-107.1); c.881G>T (p.Arg294Leu) (rs587776397) in six patients but no controls; and c.880C>T (p.Arg294Cys) (rs747142328) in two patients but no controls. In addition, c.881G>A (p.Arg294His) (rs587776397) was identified in one control. These mutations were absent in a Caucasian cohort. Carriers of these mutations had normal antithrombin levels and anticoagulant activity, consistent with results obtained in a recombinant model. However, mutation carriers had a significantly increased endogenous thrombin potential. Our results suggest the existence in the Chinese population of a hotspot in SERPINC1 that significantly increases the risk of VTE by impairing the anticoagulant capacity of the hemostatic system. This effect is not revealed by current antigen or in vitro functional antithrombin assays.
| S-EPMC5663607 | biostudies-literature
Project description:Pediatric thromboembolism (≤18 years) is very rare (0.07-0.14/10,000/year) but may be more prevalent in children with severe thrombophilia (protein C, protein S or antithrombin deficiency). The aim of this study was to define the prevalence and clinical characteristics of pediatric thrombosis in subjects with inherited antithrombin deficiency. Our observational retrospective multicentric study from two countries recruited 968 patients of any age from 441 unrelated families with genetically, biochemically and functionally characterized antithrombin deficiency. Seventy-three subjects (7.5%) developed thrombosis before 19 years of age. Two high-risk periods for thrombosis were identified: adolescence (12-18 years, n=49) with thrombus localization (lower limb deep venous thrombosis or pulmonary embolism) and triggering factors common to adults (oral contraceptives, surgery or pregnancy); and the neonatal period (<30 days, n=15) with idiopathic thrombosis at unusual sites. The clinical evaluation of pediatric thrombosis in subjects with antithrombin deficiency revealed: i) a high prevalence of cerebral sinovenous thrombosis (n=13, 17.8%), mainly at young age (8 neonates and 4 children <6 years); ii) severe outcome with fatality in six cases (3 neonates, two of them homozygous for p.Leu131Phe). The majority of subjects (76.7%) carried quantitative type I deficiency. This retrospective analysis includes the largest cohort of subjects with inherited antithrombin deficiency so far and provides strong evidence for an increased risk of pediatric thrombosis associated with this thrombophilia (300-fold compared with the general population: 0.41%/year vs 0.0014%/year, respectively). Our results support testing for antithrombin deficiency in children of affected families, particularly in case of type I deficiency.
| S-EPMC6959168 | biostudies-literature
Project description:BACKGROUND:Inherited deficiency of the antithrombin (hereditary antithrombin deficiency, AT deficiency, OMIM #613118) is a relatively rare (1:2000-3000) autosomal-dominant disorder with high risk of venous thromboembolism. Mutations in the serpin family C member 1 gene (SERPINC1) can lead to Quantitative (type I) and Qualitative (type II) types of antithrombin deficiency. We describe a new genetic variant in the SERPINC1 gene and our approach to variant interpretation. CASE PRESENTATION:We observed a 29 y.o. female proband with the episode of venous thrombosis at the age of 18 and family history of thrombosis. The antithrombin level in our patient was low, 44-48% (AT deficiency type I). A new genetic variant c.662G > C (p.W221S) in the SERPINC1 gene was detected in proband and affected father but was absent in healthy sister. We used in silico tools to evaluate the possible impact of p.W221S variant on protein structure and function. In mutated SERPINC1 protein a new N-linked glycosylation site is formed, however, it is unclear if the glycosylation at 219-221 site is possible. CONCLUSION:The proband was provided with appropriate genetic counseling and referred to a hematologist. Based on all the evidence we classify the p.W221S variant as variant of unknown clinical significance. In this paper we discuss some aspects of genetic counseling, variant interpretation and thromboembolic prophilaxis.
| S-EPMC7137186 | biostudies-literature