Antithrombin deficiency and decreased protein C activity in a young man with venous thromboembolism: a case report

Abstract

Antithrombin and protein C are two crucial members in the anticoagulant system and play important roles in hemostasis. Mutations in SERPINC1 and PROC lead to deficiency or dysfunction of the two proteins, which could result in venous thromboembolism (VTE). Here, we report a Chinese 22-year-old young man who developed recurrent and serious VTE in cerebral veins, visceral veins, and deep veins of the lower extremity. Laboratory tests and direct sequencing of PROC and SERPINC1 were conducted for the patient and his family members. Coagulation tests revealed that the patient presented type I antithrombin deficiency combined with decreased protein C activity resulting from a small insertion mutation c.848_849insGATGT in SERPINC1 and a short deletion variant c.572_574delAGA in PROC. This combination of the two mutations was absent in 400 healthy subjects each from southern and northern China. Then, we summarized all the mutations of the SERPINC1 and PROC gene reported in the Chinese Han population. This study demonstrates that the combination of antithrombin deficiency and decreased protein C activity can result in severe VTE and that the coexistence of different genetic factors may increase the risk of VTE.

Keywords

antithrombin deficiency / protein C activity / mutation / variant / venous thromboembolism / anticoagulants

Introduction

Venous thromboembolism (VTE) is a multifactorial disorder resulting from the interaction between acquired and genetic factors. On one hand, major acquired factors, such as age, family history of VTE, surgery, neoplasm, pregnancy, trauma, use of oral contraceptive, and hormone replacement therapy, play crucial roles on VTE. On the other hand, hereditary factors also exert important influence on the development of VTE [¹,²]. To date, up to 17 genes have been identified as significant determinants of VTE by genetic research [³]. In general, genetic factors result in VTE in two main models: one is by functional inhibition of endogenous anticoagulants, such as deficiency or dysfunction of protein C (PC) or protein S (PS) and antithrombin (AT); the other way is by enhancement of the function of procoagulant factors, such as PT20210A and factor V Leiden [⁴].

AT is a member of the serine superfamily of protease inhibitors synthesized in the liver; this inhibitor could primarily inactivate procoagulation factors, such as factor Xa, IXa and thrombin, heparin could accelerate the rate of the interaction process [⁵]. Inherited AT deficiency is a rare, autosomal dominant disorder, which is generally classified into two types according to the plasma antigen level and activity of AT, namely, type I AT deficiency (both antigen level and activity are decreased) and type II AT deficiency (normal antigen level and decreased activity). Inherited AT deficiency is usually due to a SERPINC1 mutation, and more than 250 different mutations of the SERPINC1 gene have been described in the databases (Human Gene Mutation Database, http://www.hgmd.org). The prevalence of inherited AT deficiency in the general population is about 5 to 17 per 1000 individuals and around 1% of VTE patients [¹]. Previous studies demonstrated that individuals with inherited AT deficiency present up to 20 times higher risk of VTE than non-deficient individuals; when the inherited AT deficiency is combined with factor V Leiden or PT20210A, the incidence of VTE could further increase fivefold [⁶]. The combination of inherited AT deficiency and PC or PS deficiency could result in severe thrombosis; however, this deficiency combination is rare and has yet to be reported in Chinese patients [⁷–¹⁰]. Here, we reported a young Chinese patient suffering from multiple and recurrent VTE with inherited type I AT deficiency combined with decreased PC activity caused by SERPINC1 and PROC mutations.

Case report

The patient was a 22-year-old male living in the countryside of Huangshi City, Hubei Province, China, with no family history of VTE. He was the second son of his non-consanguineous parents and had a healthy older brother. The first episode of VTE occurred when the patient was 17 years old in 2011; he suffered a headache and fever, fell into a coma and convulsions for 2 days without any inducement, and was sent to the neurological department of our hospital. His results were as follows: prothrombin time (PT), 4.7 s; prothrombin time activity (PTA), 58.0 s; international normalized ratio (INR), 1.30; activated partial thromboplastin time (APTT), 26.8 s; and D-dimer, 2433 mg/mL. Digital subtraction angiography (DSA) results showed thrombosis in his superior sagittal sinus, transverse sinus, and sigmoid sinus. After treatment with low-molecular weight heparin, the state of the patient was reverted. Shortly after his recovery from the first episode, the patient felt pain from his lower limb; through physical examination, increased skin temperature and edema of lower limb were observed, and deep vein thrombosis in his bilateral lower extremities was diagnosed by Color Doppler Flow Imaging. Fortunately, pulmonary embolism was precluded by pulmonary artery computed tomography angiography. After treatment for another 10 days, the symptoms disappeared. At 19 years old, the patient was affected by several abdominal pain for several times, persistent vomiting, and nausea and was brought to our hospital for the third time. Thrombosis of the superior mesenteric vein and portal vein was detected using abdominal contrast-enhanced computed tomography. Immediate treatment including anticoagulation, fasting, and intravenous nutrition was carried out and continued for 10 days, and his symptoms gradually improved. Long-term warfarin anticoagulation therapy was started. The clinical condition of the patient has been favorable ever since.

Laboratory screening of VTE was performed when the patient was treated in our hospital. The activities of AT, PC, and PS were 44%, 81%, and 99%, and the antigen levels of AT, PC, and PS were 48%, 93%, and 100% (Table 1). [The activities of AT, PC, and PS were evaluated on a STA-R evolution automatic coagulation analyzer (Diagnostica Stago, France) in the Department of Clinical Laboratory of Tongji Hospital, and the antigen levels of AT, PC, and PS were tested by enzyme-linked immunosorbent assay (Elabscience Biotechnology Co. Ltd., China).] The patient showed type I AT deficiency with reduced PC activity at the lower limit of the normal range. To detect the genetic causes underlying this condition, we extracted genomic DNA of the patient and his family members from peripheral blood lymphocytes after obtaining written consent. All exons and adjacent regions of SERPINC1 and PROC were directly sequenced on ABI 3130xl capillary sequencer (Applied Biosystems, Foster City, USA) (sequences of PCR primers are displayed in Table S1). We found that a heterozygous deletion variant of PROC c.572_574delAGA and a heterozygous insertion in SERPINC1 c.848_849insGATGT coexisted in the patient. The family study demonstrated that heterozygous PROC c.572_574delAGA of the patient was inherited from his mother, heterozygous SERPINC1 c.848_849insGATGT was inherited from his father, and the older brother also carried heterozygous SERPINC1 c.848_849insGATGT mutation inherited from his father (Fig. 1). In addition, we measured the plasma activities and antigen levels of AT, PC, and PS of the patient’s parents and brother. The mother, who carried a heterozygous PROC c.572_574delAGA variant and no SERPINC1 mutation, presented normal activities and antigen levels of AT and PS and decreased PC activity. The father and brother, who both carried a heterozygous SERPINC1 c.848_849insGATGT mutation and no PROC variant, presented type I AT deficiency and had normal activities and antigen plasma levels of PC and PS, despite the absence of symptom and sign of VTE (Table 1). Other VTE risk factors, such as the evaluated factor VIII, lupus anticoagulant, and antiphospholipid antibodies, were also examined and found to be absent in the patient.

After screening SERPINC1 c.848_849insGATGT in 400 healthy Chinese individuals by direct sequencing (200 subjects from south of China and 200 subjects from north of China), individuals with SERPINC1 c.848_849insGATGT were absent in these 400 healthy controls, and 9 (2.25%) subjects carrying PROC c.572_574delAGA were identified.

Discussion

We reported the case of a severe VTE patient with coexistence of AT deficiency and decreased PC activity caused by the combination of a heterozygous nonsense mutation of SERPINC1 c.848_849insGATGT and a heterozygous deletion variant of PROC c.572_574delAGA, whose AT and PC activity was 44% and 81%, respectively. The c.848_849insGATGT of SERPINC1 was a frameshift that resulted in protein synthesis termination and the deletion of the C-terminal domain of AT, which evidently caused protein destruction and AT deficiency. The patient, his father, and his brother carried this mutation and were all diagnosed with type I AT deficiency. This mutation was also reported in another VTE individual with decreased AT activity and AT antigen level [¹¹]. This mutation was absent in not only 400 healthy controls in China but also from the 1000 Genome Project [¹²], indicating that this mutation was rare.

The PROC c.572_574delAGA variant was first reported in Japanese PC deficiency patients [¹³]. A recent study illustrated that PROC c.572_574delAGA is a common variant with a prevalence of 2.40% in Chinese Han population, conferring 2.84-fold VTE risk in Chinese [¹⁴]. The PROC c.572_574delAGA variant resulted in a small fragment deletion of exon 7 of PROC and generated a PC product with deletion at position 192 or 193. An in vitro study demonstrated that the c.572_574delAGA variant could reduce the PC anticoagulant activity [¹⁵]. Individuals who carried this variant show lower PC anticoagulant activities than non-carriers, although the PC anticoagulant activity of most carriers was still within the normal range [¹⁵]. To avoid reader confusion regarding our statements, we used Polyphen to predict the potential functional consequences of this mutation, and the bioinformatics tool also showed that c.572_574delAGA could severely influence the protein C function. In our family study, the patient and his mother, who carried the PROC c.572_574delAGA, showed lower PC activity than his father and brother (81% and 97% vs. 122% and 108%).

VTE is the result of the interaction of one or more genetic factors and environmental risk factors in affected patients. In this family study, the parents and brother of the patient who carried one variant in PROC or SERPINC1 were all asymptomatic, whereas the patient who simultaneously carried the two aforementioned variants suffered from serious recurrent VTE at an early age, indicating that the parents may present a lower risk of suffering from a thrombosis event. This situation demonstrated that the combination of AT deficiency and decreased PC activity could result in serious VTE. This finding is similar to that with the combination of AT deficiency and prothrombotic polymorphisms (FV Leiden or PT20210) found in other patients and is another good example demonstrating that the combination of different genetic risk factors could further increase VTE risk. Moreover, the reduction of hepatic antithrombin and protein C production could result in acute and severe venous thrombosis in mice models [¹⁶]. In a recent study, Zeng et al. reported nine Chinese patients who presented normal levels of AT antigen and activity and were genetically diagnosed with AT deficiency; these outcomes emphasized the function of genetic analysis in the diagnosis of AT deficiency [¹¹]. On the basis of our study, we suggested that the coexistence of different genetic factors should be considered in the preventions of VTE. Given that we only studied a single family, we were not able to estimate the gene–gene and gene–environment interaction, therefore, further studies with larger sample size should be performed.

We also reviewed the molecular background of the SERPINC1 and PROC genes in the Chinese Han population. We searched the PubMed database and WanFang Med Online for all studies that referred to mutations of SERPINC1 and PROC for the Chinese population. A total of 58 non-synonymous mutations of SERPINC1 were summarized, as shown in Fig. 2A. Among these mutations, 28 null mutations (insertion, deletion, splice site, and nonsense mutation) were included, and exon 2 and exon 5 were the hot parts of mutations. The proportion of null mutation was close to that of patients with AT deficiency in Europe (48% vs. 41%) [¹⁷]. For the PROC gene, 64 nonsynonymous mutations were found, including 12 (18%) null mutations and 52 (82%) missense mutations, and exon 9 contained the most number of mutations (Fig. 2B). The mutation lists and the detailed information of each mutation of SERPINC1 and PROC are shown in Tables S1 and S2.

Conclusions

We performed a family study and reported a young patient who suffered from VTE with type I AT deficiency and decreased PC activity. To our knowledge, this case is the first reported in Chinese with concurrently carried gene variants of SERPINC1 and PROC. Our study enriched the insights of genetic factors for VTE and will facilitate the genetic diagnosis of this disease.

References

[1] Martinelli I, De Stefano V, Mannucci PM. Inherited risk factors for venous thromboembolism. Nat Rev Cardiol 2014; 11(3): 140–156

[2] Reitsma PH, Versteeg HH, Middeldorp S. Mechanistic view of risk factors for venous thromboembolism. Arterioscler Thromb Vasc Biol 2012; 32(3): 563–568

[3] Morange PE, Suchon P, Trégouët DA. Genetics of venous thrombosis: update in 2015. Thromb Haemost 2015; 114(5): 910–919

[4] Rosendaal FR. Venous thrombosis: a multicausal disease. Lancet 1999; 353(9159): 1167–1173

[5] Roemisch J, Gray E, Hoffmann JN, Wiedermann CJ. Antithrombin: a new look at the actions of a serine protease inhibitor. Blood Coagul Fibrinolysis 2002; 13(8): 657–670

[6] van Boven HH, Vandenbroucke JP, Briët E, Rosendaal FR. Gene-gene and gene-environment interactions determine risk of thrombosis in families with inherited antithrombin deficiency. Blood 1999; 94(8): 2590–2594

[7] Jobin F, Vu L, Lessard M. Two cases of inherited triple deficiency in a large kindred with thrombotic diathesis and deficiencies of antithrombin III, heparin cofactor II, protein C and protein S. Thromb Haemost 1991; 66(3): 295–299

[8] Sansores-García L, Majluf-Cruz A. Arterial and venous thrombosis associated to combined deficiency of protein C and antithrombin III. Am J Hematol 1998; 57(2): 182–183

[9] Bowen D, Dasani H, Yung B, Bloom A. Deep venous thrombosis and pulmonary embolism in a patient with type III von Willebrand’s disease, protein C and antithrombin III deficiency. Br J Haematol 1992; 81(3): 446–447

[10] Gouault-Heilmann M, Quetin P, Dreyfus M, Gandrille S, Emmerich J, Leroy-Matheron C, Guesnu M. Massive thrombosis of venous cerebral sinuses in a 2-year-old boy with a combined inherited deficiency of antithrombin III and protein C. Thromb Haemost 1994; 72(5): 782–783

[11] Zeng W, Tang L, Jian XR, Li YQ, Guo T, Wang QY, Liu H, Wu YY, Cheng ZP, Hu B, Lu X, Yu JM, Deng J, Wang HF, Sun CY, Yang Y, Hu Y. Genetic analysis should be included in clinical practice when screening for antithrombin deficiency. Thromb Haemost 2015; 113(2): 262–271

[12] 1000 Genomes Project Consortium, Abecasis GR, Altshuler D, Auton A, Brooks LD, Durbin RM, Gibbs RA, Hurles ME, McVean GA. A map of human genome variation from population-scale sequencing. Nature 2010; 467( 7319): 1061–1073

[13] Miyata T, Sakata T, Yasumuro Y, Okamura T, Katsumi A, Saito H, Abe T, Shirahata A, Sakai M, Kato H. Genetic analysis of protein C deficiency in nineteen Japanese families: five recurrent defects can explain half of the deficiencies. Thromb Res 1998; 92(4): 181–187

[14] Tang L, Wang HF, Lu X, Jian XR, Jin B, Zheng H, Li YQ, Wang QY, Wu TC, Guo H, Liu H, Guo T, Yu JM, Yang R, Yang Y, Hu Y. Common genetic risk factors for venous thrombosis in the Chinese population. Am J Hum Genet 2013; 92(2): 177–187

[15] Tang L, Lu X, Yu JM, Wang QY, Yang R, Guo T, Mei H, Hu Y. PROC c.574_576del polymorphism: a common genetic risk factor for venous thrombosis in the Chinese population. J Thromb Haemost 2012; 10(10): 2019–2026

[16] Safdar H, Cheung KL, Salvatori D, Versteeg HH, Laghmani H, Wagenaar GT, Reitsma PH, van Vlijmen BJ. Acute and severe coagulopathy in adult mice following silencing of hepatic antithrombin and protein C production. Blood 2013; 121(21): 4413–4416

[17] Luxembourg B, Delev D, Geisen C, Spannagl M, Krause M, Miesbach W, Heller C, Bergmann F, Schmeink U, Grossmann R, Lindhoff-Last E, Seifried E, Oldenburg J, Pavlova A. Molecular basis of antithrombin deficiency. Thromb Haemost 2011; 105(4): 635–646

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