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Clinical and genetic features of protein C deficiency in 23 unrelated Chinese patients
Blood Cells, Molecules, and Diseases 50 (2013) 53–58
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Clinical and genetic features of protein C deficiency in 23 unrelated Chinese patients☆,☆☆ Qiulan Ding a, 1, Wei Shen b, 1, Xu Ye c, Yingting Wu d, Xuefeng Wang a,⁎, Hongli Wang d a
Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Department of Laboratory Medicine, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Department of Hematology, Second affiliated Hospital of Guangzhou Medical University, Guangzhou, China d State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China b c
a r t i c l e
i n f o
Article history: Submitted 24 July 2012 Revised 8 August 2012 Available online 27 August 2012 (Communicated by U. Seligsohn, Ph.D., 9 August 2012) Keywords: Gene mutation Protein C deficiency Thromboembolism Thrombophilia
a b s t r a c t In this study, we investigated the clinical and genetic features of protein C deficiency in the Chinese population. A total of 23 symptomatic patients with protein C deficiency were identified by thrombophilic assays. Detailed clinical data about the patients with respect to their personal and family history of venous thromboembolism (VTE) were collected. Mutational analysis was then performed by direct sequencing of the protein C gene (PROC) in the patients and their family members. Of the 23 patients, 30.4% (7/23) had additional risk factors, 51.2% (12/23) suffered from recurrent thrombotic episodes, and 50.0% (6/12) of the patients with recurrent thrombosis had more than one heterozygous mutation in PROC itself or combined with protein S gene (PROS). The sex distribution of male:female was 19:4 in the 23 symptomatic patients and 10:2 in the 12 recurrent patients. Almost all patients (22/23) had lower extremity deep vein thrombosis (DVT) and one had pulmonary embolism (PE) only. A total of 15 different causative mutations were identified from the 23 subjects with 6 (40.0%) of the mutations being novel. Among the mutations identified, the Arg147Trp substitution was hotspot mutation in the Chinese population with a high frequency of 43.5%. Our finding suggests that complex genotypes of PROC or combined with protein S deficiency are primarily responsible for an increased risk of recurrent VTE. Our data further provides a framework for correlating the clinical pathogenesis of protein C deficiency to ethnic backgrounds in the Chinese population. © 2012 Elsevier Inc. All rights reserved.
Introduction Venous thromboembolism (VTE) is a common multifactorial disease resulting from the interaction of genetic and environmental risk factors. Genetic abnormalities of proteins involving the coagulation pathway that lead to hypercoagulability have been found in subjects suffering from thrombophilic disease. Factor V Leiden (FV Leiden) and prothrombin G20210A gene mutations are highly prevalent in the Caucasian population but are almost non-existent among the Asian population [1]. In contrast, a higher prevalence of protein Abbreviations: VTE, venous thromboembolism; DVT, deep venous thrombosis; PE, pulmonary embolism; PVT, portal venous thrombosis; MVT, mesenteric venous thrombosis; AVT, axillary vein thrombosis; PROC, protein C gene; PROS, protein S gene; PC:A, activity of protein C; PC:Ag, antigen of protein C. ☆ This study was partially supported by Shanghai Municipal Health Bureau of young scientific research project (2011Y195). ☆☆ Conflicts of interest and sources of funding: The authors stated that they had no interests which might be perceived as posing a conflict or bias. ⁎ Corresponding author at: Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, No. 197 Ruijin Second Road, Shanghai 200025, China. Fax: +86 21 64333548. E-mail address: [email protected] (X. Wang). 1 These authors made equal contributions to this work. 1079-9796/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bcmd.2012.08.004
S and protein C deficiency has been reported in the Asian population [2–5]. Two studies have reported that protein C and protein S deficiencies are the most important risk factors associated with thrombosis in Taiwanese and Japanese populations [3,4]. Protein C is a vitamin K-dependent serine protease zymogen synthesized in the liver. It is activated by thrombin in complex with thrombomodulin on the surface of endothelial cells. The activated protein C (APC), in complex with its cofactor protein S, inactivates procoagulant cofactors Va and VIIIa, thereby down-regulating thrombin generation in coagulation cascade. Protein C deficiency has an autosomal dominant pattern of inheritance and is associated with an increased risk of VTE. Heterozygosity for the disease has been shown to increase the risk of venous thrombosis by 5- to 10-fold [6]. Homozygosity and compound heterozygosity of protein C are associated with neonatal purpura fulminans or severe thromboembolic complications after birth. In rare cases, thrombotic episodes develop in childhood or adulthood. The prevalence of protein C deficiency defined by plasma levels is between 0.2% and 0.5% of the healthy population [7,8]. Most of the affected individuals remain asymptomatic throughout their life and 2–5% present clinically symptomatic protein C deficiencies, suggesting that protein C deficiency alone is a relatively low risk factor for thrombosis [9,10]. In Caucasians,
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the presence of the two common mutations, factor V Leiden and prothrombin G20210A, has been associated with a more severe phenotype in some protein C deficient patients [11,12]. However, available data are scarce regarding which kinds of additional genetic and environmental risk factors contribute to the onset of first-time and recurrent thrombotic episodes in patients with protein C deficiency in the Asian population. The human protein C gene (PROC) is localized in chromosome 2q13–q14 and comprises nine exons spanning more than 11 kb of genomic DNA. More than 300 mutations have been reported so far, and the spectrum of PROC mutations has been reported in several studies. Most reports are restricted to Western populations with only a few having been reported in the Asian population [11,13–15]. Results of these studies have indicated that the mutation profile of PROC is significantly influenced by ethnic background, thus, some mutations (Arg230Cys, Arg178Trp, Gln132X, Val297Met and Pro168Leu) are commonly found in the Caucasian population, while the others (Phe139Val, Arg169Trp, Val297Met, Met364Ile, and G8857del) are recurrent defects in the Japanese population. So far, few studies have been published on the spectrum of PROC defects and its thrombotic manifestation in patients with protein C deficiency in mainland China. In this study, we addressed this question by analyzing the molecular defects of the PROC in the 23 symptomatic subjects. Our results demonstrate a correlation between the ethnic background and the occurrence of thrombotic episodes stemming from protein C deficiency in the Chinese population. Materials and methods Patients This study was approved by the Ethics Committee of Ruijin Hospital, Shanghai Jiaotong University School of Medicine. Beginning in January 2002–January 2012, 28 unrelated patients with first-time or recurrent VTE episodes were referred to our thrombosis center due to their predisposition to the risk factors for VTE and finally were found that they were with protein C deficiency. All the index subjects were interviewed with respect to their medical history. The diagnosis of VTE and the presence of acquired risk factors, including immobilization, fractures, pregnancy, puerperium, oral contraceptives, hormone replacement, surgery, and malignancy, at the time of all episodes of VTE were recorded. The VTE diagnosis was based only on the results of objective investigations employing the following approaches: compression or color Doppler ultrasonography was used to diagnose deep venous thrombosis (DVT) and abdominal venous thrombosis; a high-probability ventilation–perfusion scan, pulmonary angiography, or computed tomography (CT) was used to diagnose pulmonary embolism (PE); magnetic resonance venography (MRV) or CT was used to diagnose occlusion of cerebral or abdominal veins. A detailed family history was obtained from the family members with special emphasis on the occurrence of prior VTE events. Hemostatic assays Venous blood samples from the patients and their family members were collected in 0.109 mmol/L sodium citrate after informed consent was obtained. Tests for thrombophilia including antithrombin (AT), protein C, protein S, plasminogen (PLG), tissue plasminogen activator (t-PA), lupus anticoagulant (LA), anticardiolipin antibody (ACA), anti-β2 glycoprotein I (anti-β2GPI), fibrinogen (Fg), and total homocysteine (Hcy). The plasma levels of ACA and anti-β2GPI (Euroimmun, Lübeck, Germany), and t-PA (Instrumentation Laboratory, Milan, Italy) were detected using enzyme-linked immunosorbent assays (ELISA) according to the manufacturers' instructions. LA was detected using a diluted viper venom time (DVVT) assay (Instrumentation Laboratory). The Hcy levels were determined using the AxSYM homocysteine kit
(Abbott, Lake County, IL, USA) based on a fluorescence polarization immunoassay (FPIA). The functional and antigenic fibrinogen levels were detected using the Clauss method on a Sysmex CA7000 analyzer (Sysmex Corporation, Tokyo, Japan) and immunoturbidimetric assay, respectively. The activities of protein C, AT, and PLG (PC:A, AT:A, and PLG:A) were analyzed using chromogenic substrate methods (Instrumentation Laboratory). The activity of free protein S (FPS:A) was assayed using a clotting method based on prothrombin time (Instrumentation Laboratory). Free protein S antigen (FPS:Ag) was measured with a polyclonal anti-human protein S antibody using the ZYMUTEST free protein S kit (Hyphen BioMed, Neuville-Sur-Oise, France). Protein C antigen (PC:Ag) was determined using a sandwich ELISA method with a rabbit anti-human protein C polyclonal antibody (Dako, Glostrup, Denmark) as a capture antibody and a horseradish peroxidase (HRP) conjugated antibody as a detection antibody. Protein C deficiency was diagnosed only in cases where the activity of protein C (PC:A) was lower than the normal level and both liver and renal functions were normal. Genetic analysis of PROC Genomic DNA was extracted from the peripheral blood leukocytes using the QIAamp DNA mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Mutations in the PROC and protein S gene (PROS) were identified as previously described [5]. The detected mutations were confirmed by reverse sequencing and were verified using a second amplicon. To rule out polymorphisms, the novel missense mutations were screened in 50 normal individuals. Only the corresponding sequence was amplified and sequenced in family members. The genetic alterations were reported according to both the standard international nomenclature guidelines recommended by the Human Genome Variation Society (HGVS; http://www.hgvs.org/mutnomen/recs.html), with nucleotide + 1 as the A of the ATG translation initiation codon and Foster's numbering system. The genomic DNA (GenBank: NM_000312.2) and cDNA (GenBank: P04070) sequences of PROC were used as references. Results Association between the genetic variants of PROC and onset of thrombotic episodes A total of 28 patients were diagnosed as having protein C deficiency, 2 patients had combined protein C and protein S deficiency, defined by the levels of PC:A and PS:A in their plasma. Genetic defects in the PROC were identified in 23 unrelated patients. No PROC mutation was identified in the other five patients, who were diagnosed with acquired protein C deficiency due to liver diseases. One heterozygous mutation in the PROS was found in one of the two patients with combined protein C and protein S deficiency, but no mutation was identified in the other patient with an FPS:A level of 18.0% and a FPS:Ag level of 23.7%, respectively. The subject's mother had a similarly low FPS level, suggesting that the protein S deficiency may be hereditary in the family. The characteristics of the genetic defects and the onset of thrombotic episodes in the 23 unrelated symptomatic patients with protein C deficiency are shown in Table 1. Of the 23 symptomatic subjects, 30.4% (7/23) had additional risk factors. Of the seven patients, four presented the acquired risk factors at the time of onset of VTE: surgery in patient 7 and patient 15; puerperium in patient 13; and the thrombotic complication related to the use of a permanent filter and/or advanced age in patient 10. Three patients exhibited other genetic risk factors: an elevated homocyteine level in patient 21, protein C and protein S deficiency coexisted in patients 5 and 6. Results of all the other thrombophilic assays were normal in the 23 symptomatic patients (data not shown). Twelve patients (51.2%) suffered from recurrent thrombotic episodes, six of
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Table 1 Characteristics of the genetic defects and onset of thrombotic episodes in 23 unrelated Chinese patients with protein C deficiency. No. of patients
Age/ sex
Activity (%)
Antigen (%)
Nucleotide variation (DNA)
Amino acid substitution†
1
16/M
21.0
20.0
g.26T>C g.8599C>T
2
20/M
19.0
42.0
3
28/M
21.0
18.4
4
27/M
39.0
42.1
5
30/M
36.0 18.0a 57.0 23.7.b
6
24/M
46.0 19.5a 64.4 20.9b
7
47/M
30.0
48.4
8
7/M
26.0
46.1
p.Leu8(−34)Pro p.Thr337 (295) Ile g.1474G>A p.Glu71 (29) Lys g.6152C>T p.Arg189 (147) Trp p.Arg220 (178) g.6245C>T g.8478G>C Trp p.Asp297 (255) His g.5498C>T p.Arg57 (15) Trp g.6152C>T p.Arg189 (147) Trp p.Leu8 (−34) g.26T>C Pro# g.7212 C>T g.6161-6163delAAG p.Ala251 (209) Val# p.Lys192 (150) del g.6152C>T p.Arg189 (147) c.1680T>Ac Trp p.Tyr560X (Tyr519X)d g.6152C>T p.Arg189 (147) g.6245C>T Trp# p.Arg220(178) Trp# g.5498 C>T p.Arg57(15)Trp
9
37/M
64.0
80.9
g.3426G>T
10
84/F
54.6
11
24/F
65.9
76.5
12
24/F
38.6
45.3
13
33/F
63.9
71.8
14
19/M
69.4
74.9
15
68/M
46.8
67.2
16
28/M
63.9
64
17
28/M
68.7
68.2
18
24/M
55.0
ND
19
64/M
65.0
ND
20
69/M
45.6
71.0
21
23/M
65.3
75.6
22
42/M
63.0
ND
23
40/M
49.0
43.2
p.Asp170(128) Tyr g.3442G>A p.Cys175(133) Tyr g.6152C>T p.Arg189(147) Trp g.6152C>T p.Arg189(147) Trp g.6152C>T p.Arg189(147) Trp g.6152C>T p.Arg189(147) Trp g.6152C>T p.Arg189(147) Trp g.6152C>T p.Arg189(147) Trp g.6161-6163delAAG p.Lys192(150) del g.8478G>C p.Asp297(255) His g.8478G>C p.Asp297(255) His g.8746T>C p.Leu386(344) Pro g.8763G>A p.Gly392(350) Arg g.8807G>A p.Met406(364) Ile g.8831G>A p.Trp414(372)X
Thrombotic episodes (onset age)
Thrombophilic factors
Thrombotic episodes (family member)
DVT (14), DVT (15)
PE (19)
DVT, PVT (father with p.Glu29Lys)
DVT(21), MVT(21), DVT (23)
DVT (mother with p.Asp255His)
DVT(25), DVT(26)
DVT (28y), DVT (30)
Protein S deficiency
DVT (mother with p.Lys150del combined with protein S deficiency), MVT (brother with the double mutations)
PE (22), DVT(22), DVT (24)
Protein S deficiency
DVT (father with p.Tyr519X in PROS)
DVT (40y), DVT (43)
Cholecystectomy
DVT (brother with the double mutations)
DVT(4), DVT(7), AVT (7) DVT (37) DVT(79), DVT(83)
Permanent filter, advanced age
DVT (21) DVT (23), DVT (24) DVT (33)
Puerperium
DVT (19) Both DVT and PE (68)
Cholecystectomy
DVT (28) DVT (27) DVT (23) DVT (62), DVT (63) DVT (69) DVT (23)
Hyperhomocysteinemia
DVT (37), DVT (41)
DVT (mother with p.Met364Ile)
DVT (39), DVT (40)
DVT (sister with p.Trp372X)
Reference range: PC:A: 70–140%; PC:Ag: 70–130%; FPS:A: 63–135%; FPS: Ag: 60–150%. † : The numbering of changed amino acid residue was given according to the beginning from the mature protein C or protein S within parentheses; #: double mutations. DVT: deep venous thrombosis (indicating the low extremity here); PE: pulmonary embolism; PVT: portal venous thrombosis; MVT: mesenteric venous thrombosis; AVT: axillary vein thrombosis. ND: no detection. a FPS:A. b FPS: Ag. c PS cDNA. d PS amino acid substitution.
these patients (patient 1, patients 3–7) had more than one heterozygous mutation in PROC and PROS: patient 1 and patients 3–4 carried compound heterozygous PROC mutations with the ranges of protein C levels in activity from 21.0% to 39.0% and antigen from 18.4% to 42.1%; patient 5 had a PC:A of 36.0% and a PC:Ag of 57.0%, a FPS:A
of 18.0% and a FPS:Ag of 23.7%, this patient also carried triple heterozygous PROC variants, but no genetic defect was found in the PROS. A heterozygous mutation was identified in both the PROC and PROS in patient 6. Patient 7 may exhibit two mutations on the same allele, predicted by his brother, who inherited the same mutations, and his
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Table 2 Genetic defects in the PROC identified in 23 unrelated Chinese patients with protein C deficiency. Mutations
No. of families
Leu8 (−34) Pro Arg57 (15) Trp Glu71 (29) Lys Asp170 (128) Tyr Cys175 (133) Tyr Arg189 (147) Trp Arg220 (178) Trp Ala251 (209) Val Asp297 (255) His Thr337 (295) Ile Leu386 (344) Pro Gly392 (350) Arg Met406 (364) Ile Trp414 (372) X Lys192 (150) del Total
2 2 1 1 1 10 2 1 3 1 1 1 1 1 2
Reported in Asia
Reported in worldwide
Reported in this study
+ + + + +
Other reported mutations in the same residue Arg15Gln, Gly Glu29X
+ + +
Arg178Gln,Pro
+ + + + + + + 5
4
Trp372Arg
6
The numbering of changed amino acid residue was given according to Foster's numbering (beginning from the mature protein C) within parentheses.
sister, who had normal PROC. Only one heterozygous PROC mutation was identified in each of the remaining six subjects (patients 8, 10, 12, 19, 22 and 23) with recurrent VTE events. Patient 8, with low levels of PC:A and PC:Ag (26.0% and 46.1%, respectively), developed recurrent DVT at a very early age. Recurrent DVT was associated with advanced age in two subjects (patients 10 and 15). No predisposing risk factors were detected in the other three patients other than the heterozygous protein C deficiency. At least one family member had VTE in 7 of the 23 unrelated families; 6 of these occurred in the pedigrees with patients having recurrent DVT. Considering the localization of the thromboembolic events, all of the other 22 subjects (95.7%) had lower-extremity DVT except for patient 2, who had compound heterozygous mutations in PROC and manifested only PE. Of the 22 subjects, patients 6 and 15 also had PE; patient 3 had both DVT and mesenteric venous thrombosis (MVT); and patient 8 had an axillary vein thrombosis (AVT) together with DVT. The mean age at the time of the first thrombotic episode was 33.1 years (range: 4 to 79 years) in the 23 symptomatic patients. In patients 1–7 who had more than one variant in the PROC and PROS, the average age of the first episode of VTE was 24.1 years. Four subjects (patients 1, 2, 8 and 14) experienced VTE before the age of 20. Sex distribution of male:female was 19:4 in the 23 symptomatic patients and 10:2 in the 12 recurrent patients, respectively, suggesting that of those with protein C deficiency, male may have a much higher frequency of developing first and recurrent VTE than does female in Chinese population.
Characteristics of genetic defects of PROC in the 23 unrelated patients In this study, we reported the spectrum of the PROC mutations in 23 subjects with protein C deficiency. A total of 15 different causative mutations were identified including 13 missense mutations (86.7%), one nonsense mutation (6.7%), and one in-frame deletion (6.7%). Among the 15 identified mutations, 6 (40.0%) had not been mentioned either in the database or in the literature; 4 (26.7%) had been reported worldwide and 5 (33.3%) only in Asia. Of the 6 novel mutations, 5 were missense mutations, including Glu29Lys in the gamma-carboxyglutamic (Gla) domain, Asp128Tyr and Cys133 Tyr in the epidermal growth factor (EGF)-like domain, Thr295Ile, Thr315Ala, Leu344Pro in the serine protease domain, and the last one was a nonsense mutation (Trp372X) located in the serine protease domain (Table 2).
While six mutations were found in more than one patient, the Arg147Trp mutation occurred 10 times with a frequency of 43.5% (10/23) and the Asp255His occurred three times (13.0%). All remaining four mutations (Leu-34Pro, Arg15Trp, Lys150del and Arg178Trp) occurred two times. The high recurrence incidence of the Arg147Trp mutation suggested but did not prove a founder effect in the Chinese population (Table 2). Discussion This is the first report that describes both the clinical and genetic features of protein C deficiency in 23 unrelated subjects in the Chinese population. It also outlines the relationship among the genetic alterations of PROC with respect to the onset of thrombotic episodes. Heterozygous protein C deficiency is inherited in an autosomal dominant fashion, and its frequency in the Chinese population is approximately 0.29% [5]. Several studies have suggested that additional genetic risk factors can coexist and contribute to thrombotic risk in patients with protein C deficiency. Due to a high prevalence of FV Leiden in a general Caucasian population (prevalence, 2–7%), and in patients (prevalence, 20–40%) with thrombosis [16], the combination of protein C deficiency plus FV Leiden is relatively common. A previous report found that 14% of a group of 113 unrelated protein C heterozygotes also had the FV Leiden [11]. In another report, 73% of family members who were heterozygous for both protein C deficiency and FV Leiden mutation manifested thrombosis. In contrast, 31% and 13% of family members having either the protein C deficiency or FV Leiden mutation, respectively, had experienced a thrombotic episode [12], indicating that protein C deficiency combined with FV Leiden mutation presents an increased risk of thrombosis. However, few cases of FV Leiden mutation have been identified in either Asian or African populations. In this study, of the 23 patients with symptomatic protein C deficiency, 30.4% (7/23) had additional risk factors. Four patients presented acquired risk factors, and three patients exhibited other genetic risk factors. These results are consistent with the previous report that the initial episode of VTE in patients with protein C deficiency is associated with other genetic and acquired risk factors in approximately 30% of the subjects [17]. Combined protein C and protein S defects are rare and have been reported in association with ischemic strokes, multiple aortic thrombosis, mesenteric artery thrombosis, DVT, intracardiac multichamber thrombi, avascular necrosis, and portal vein thrombosis (PVT) [18–21]. The prevalence of thrombotic events among patients with combined protein C and
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protein S defect has not been reported. Here we have identified two subjects with combined protein C and protein S deficiency, who had their first thrombotic events in their early twenties. In addition, at least one family member suffered from VTE in each of the two pedigrees. We predict that, unlike in Caucasians, coexisting protein S deficiency rather than FV Leiden mutation may contribute to a more severe risk of thrombosis in subjects with protein C deficiency in the Chinese population. Surprisingly, in one case identified among the 3493 Chinese adult blood donors and healthy individuals, a 40-year-old male with coexisting protein C and protein S deficiency had never experienced thrombotic events nor had his family members, indicating that an asymptomatic carrier of both heterozygous protein C and protein S deficiency can exist as a normal individual [5]. Severe homozygous or compound heterozygous protein C deficiency occurs in approximately 1 in 500,000 to 1 in 750,000 live births. Homozygous and compound heterozygous protein C deficiencies are classically associated with neonatal purpura fulminans (NPF), massive thrombosis, and disseminated intravascular coagulation (DIC) at birth. Occasionally, patients with low levels of PC:A present with VTE in childhood or adolescence. In this study, we found a very high frequency of complex genotypes of PROC in the 23 symptomatic subjects and in the 12 recurrent VTE subjects, including one double heterozygous, four compound heterozygous, and one triple heterozygous mutations. Of these, five had recurrent VTE events and one subject experienced only a PE episode. One of the possible reasons may be that most patients in our study were recruited consecutively among outpatients who had idiopathic first and recurrent episodes of VTE at a young age, without definite acquired risk factors. Therefore, the onset of VTE tends to be provoked by inherited thrombophilic defects. Thus, we predict that complex genotypes of PROC may play an important role in the protein C deficient patients with recurrent VTE in the Chinese population. In our report, 95.7% of symptomatic protein C deficient subjects (22/23) had thrombosis in the deep veins of the lower extremities and one only had PE. Co-occurrence of thrombotic events was recorded as well: PE in patients 6 and 15 (9.1%), MVT in patient 3 (4.5%), and AVT in patient 8 (4.5%). The much higher frequency of lower extremity DVT was consistent with previous reports that the co-occurrence of other types of VTE is low in patients with protein C deficiency [22,23]. Of the 23 symptomatic patients, the average age at onset of the first thrombotic episode was 33.1 years, which is consistent with a previous study which demonstrated that in heterozygous individuals thrombotic episodes occur at age 30–40 years and is rare before the age of 20 years [22]. Whereas in the six patients with complex genotypes of PROC (patient 1–5, 7), the average age of the first onset of VTE was earlier (24.5 years). As expected for an autosomal genetic disorder, the prevalence of hereditary protein C deficiency is similar between males and females. However, there is firm evidence that the male sex in itself is an important risk factor for VTE. Males not only have a higher risk of a first VTE (1.4-fold) than females, but they also have a 2.5-fold higher risk of thrombotic recurrence than do females [24,25]. Moreover, the idiopathic first thrombotic events were more common in males, while first thrombotic events associated with special risk factors (e.g., oral contraceptive use, pregnancy, the postpartum state, and estrogen-containing hormonal therapy) were reported almost five times more often in women. We found a high male:female ratio in the symptomatic subjects (19:4) and recurrent subjects (10:2) but a low ratio in the 10 healthy subjects (3:7) [5], revealing that male patients with protein C deficiency have a much higher frequency of venous thromboembolic events than do females. A clear explanation for this finding is unknown and further investigation of the interaction between the sex of the subject and protein C deficiency contributing to VTE may need to be conducted. In China, only 2.22% of women of childbearing age take an oral contraceptive, whereas most of them choose an intrauterine device (49.13%) or surgical sterilization (36.25%) for birth control, according to the
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statistics of the State Family Planning Commission in 2009. This may reduce the oral contraceptive use associated with VTE occurrence in the individuals with protein C deficiency in the Chinese population. We identified a total of 15 different causative mutations of PROC in the 23 subjects with protein C deficiency, including 13 missense mutations (86.7%), one nonsense mutation (6.7%), and one in-frame deletion (6.7%). Of these, 40.0% (6/15) were novel mutations, indicating that the mutation spectrum of PROC is heterogeneous in the Chinese population. In contrast, we found one ethnic-specific mutation: the Arg147Trp mutation which occurred 10 times (43.5%) in 23 unrelated subjects (Table 2). The Arg147Trp mutation was first identified in an American of Hawaiian descent [26], and the high prevalence of heterozygotes (0.85%) for this mutation has been reported for the normal Chinese population. In addition, Arg147Trp was also the most common genetic defect among symptomatic patients in the Taiwanese population, and more than half of the symptomatic patients had the mutation in combination with other protein C or protein S deficiency and/or environmentally precipitating factors [27]. Similar results were found in our study: 60% of patients (6/10) had a similar combination of risk factors to those reported in the previous study, indicating that Arg147Trp mutation is associated with VTE but is a relatively weak risk factor. To sum up our previously reported mutations of PROC in 13 unrelated subjects [5,28,29], we found two significant ethnicity-specific mutations: Arg147Trp and Lys150del occurred 10 times (27.8%) and 5 times (13.9%), respectively, in the 36 unrelated subjects. The Lys150del was first reported in the Japanese population [13], and a subsequent study showed that it was one of the most prevalent mutations in the Japanese population [3]. Considering their high frequencies, further biochemical and functional analysis of these two hotspot protein C mutations (Arg147Trp and Lys150del) in the Chinese population is worth pursuing. In conclusion, we have demonstrated that the clinical and genetic features of protein C deficiency have some unique differences among Chinese, Caucasian, and perhaps even other Asian populations. Unlike the Caucasian population, complex genotypes of PROC or combination of protein C and protein S deficiency are mainly responsible for an increased risk of VTE, especially for recurrent VTE. For the first time, we have demonstrated that male patients with protein C deficiency have a much higher frequency of VTE than do females in the Chinese population. Our results may explain the molecular basis for the pathogenesis of protein C deficiency according to ethnic background and may help to construct pathogenesis-based prophylaxis and treatment strategies for VTE in the Chinese population. Acknowledgments The authors wish to thank Audrey Rezaie for proofreading the manuscript and thank patients and their family members for their participation in this study. References [1] M. Margaglione, E. Grandone, Population genetics of venous thromboembolism. A narrative review, Thromb. Haemost. 105 (2011) 221–231. [2] P. Angchaisuksiri, Venous thromboembolism in Asia—an unrecognised and under-treated problem? Thromb. Haemost. 106 (2011) 585–590. [3] T. Miyata, Y. Sato, J. Ishikawa, et al., Prevalence of genetic mutations in protein S, protein C and antithrombin genes in Japanese patients with deep vein thrombosis, Thromb. Res. 124 (2009) 14–18. [4] M.C. Shen, J.S. Lin, W. Tsay, Protein C and protein S deficiencies are the most important risk factors associated with thrombosis in Chinese venous thrombophilic patients in Taiwan, Thromb. Res. 99 (2000) 447–452. [5] T. Zhu, Q. Ding, X. Bai, et al., Normal ranges and genetic variants of antithrombin, protein C and protein S in the general Chinese population. Results of the Chinese hemostasis investigation on natural anticoagulants study I group, Haematologica 96 (2011) 1033–1040. [6] B. Dahlback, The protein C anticoagulant system: inherited defects as basis for venous thrombosis, Thromb. Res. 77 (1995) 1–43. [7] J. Miletich, L. Sherman, G. Broze Jr., Absence of thrombosis in subjects with heterozygous protein C deficiency, N. Engl. J. Med. 317 (1987) 991–996.
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[8] R.C. Tait, I.D. Walker, P.H. Reitsma, et al., Prevalence of protein C deficiency in the healthy population, Thromb. Haemost. 73 (1995) 87–93. [9] C.L. Gladson, I. Scharrer, V. Hach, et al., The frequency of type I heterozygous protein S and protein C deficiency in 141 unrelated young patients with venous thrombosis, Thromb. Haemost. 59 (1988) 18–22. [10] J. Mateo, A. Oliver, M. Borrell, et al., Laboratory evaluation and clinical characteristics of 2,132 consecutive unselected patients with venous thromboembolism—results of the Spanish Multicentric Study on Thrombophilia (EMET-Study), Thromb. Haemost. 77 (1997) 444–451. [11] S. Gandrille, J.S. Greengard, M. Alhenc-Gelas, et al., Incidence of activated protein C resistance caused by the ARG 506 GLN mutation in factor V in 113 unrelated symptomatic protein C-deficient patients. The French Network on the behalf of INSERM, Blood 86 (1995) 219–224. [12] B.P. Koeleman, P.H. Reitsma, C.F. Allaart, et al., Activated protein C resistance as an additional risk factor for thrombosis in protein C-deficient families, Blood 84 (1994) 1031–1035. [13] T. Miyata, T. Sakata, Y. Yasumuro, et al., Genetic analysis of protein C deficiency in nineteen Japanese families: five recurrent defects can explain half of the deficiencies, Thromb. Res. 92 (1998) 181–187. [14] P.H. Reitsma, S.R. Poort, C.F. Allaart, et al., The spectrum of genetic defects in a panel of 40 Dutch families with symptomatic protein C deficiency type I: heterogeneity and founder effects, Blood 78 (1991) 890–894. [15] M.C. Shen, J.S. Lin, W. Tsay, High prevalence of antithrombin III, protein C and protein S deficiency, but no factor V Leiden mutation in venous thrombophilic Chinese patients in Taiwan, Thromb. Res. 87 (1997) 377–385. [16] D.C. Rees, M. Cox, J.B. Clegg, World distribution of factor V Leiden, Lancet 346 (1995) 1133–1134. [17] I. Pabinger, B. Schneider, Thrombotic risk in hereditary antithrombin III, protein C, or protein S deficiency. A cooperative, retrospective study. Gesellschaft fur Thromboseund Hamostaseforschung (GTH) Study Group on Natural Inhibitors, Arterioscler. Thromb. Vasc. Biol. 16 (1996) 742–748.
[18] M. Hayashida, H. Yamada, S. Yamazaki, et al., Combined protein C and protein S deficiency in a family with repetitive thromboembolism and segregated gene mutations, Intern. Med. 42 (2003) 268–272. [19] S. Hwang, D.Y. Kim, M. Kim, et al., Deficiencies in proteins C and S in a patient with idiopathic portal hypertension accompanied by portal vein thrombosis, Korean J. Hepatol. 16 (2010) 176–181. [20] M.J. Kim, S.H. Hur, Y.S. Lee, et al., Intracardiac multichamber thrombi in a patient with combined protein C and protein S deficiencies, Int. J. Cardiol. 100 (2005) 505–506. [21] M. Nandi, R. Mondal, Avascular necrosis of hip following combined protein C and protein S deficiency, Indian Pediatr. 48 (2011) 992. [22] R.P. Lensen, F.R. Rosendaal, T. Koster, et al., Apparent different thrombotic tendency in patients with factor V Leiden and protein C deficiency due to selection of patients, Blood 88 (1996) 4205–4208. [23] E.M. Wysokinska, W.E. Wysokinski, R.D. Brown, et al., Thrombophilia differences in cerebral venous sinus and lower extremity deep venous thrombosis, Neurology 70 (2008) 627–633. [24] S.C. Christiansen, S.C. Cannegieter, T. Koster, et al., Thrombophilia, clinical factors, and recurrent venous thrombotic events, JAMA 293 (2005) 2352–2361. [25] P.A. Kyrle, E. Minar, C. Bialonczyk, et al., The risk of recurrent venous thromboembolism in men and women, N. Engl. J. Med. 350 (2004) 2558–2563. [26] W. Tsay, J.S. Greengard, R.R. Montgomery, et al., Genetic mutations in ten unrelated American patients with symptomatic type 1 protein C deficiency, Blood Coagul. Fibrinolysis 4 (1993) 791–796. [27] W. Tsay, M.C. Shen, R147W mutation of PROC gene is common in venous thrombotic patients in Taiwanese Chinese, Am. J. Hematol. 76 (2004) 8–13. [28] T. Yu, J. Dai, H. Liu, et al., Homozygous protein C deficiency with late onset venous thrombosis: identification and in vitro expression study of a novel Pro275Ser mutation, Pathology 44 (2012) 348–353. [29] R.F. Zhou, X.H. Cai, S. Xie, et al., Molecular mechanism for hereditary protein C deficiency in two Chinese families with thrombosis, J. Thromb. Haemost. 4 (2006) 1154–1156.
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Blood Cells, Molecules, and Diseases journal homepage: www.elsevier.com/locate/bcmd
Clinical and genetic features of protein C deficiency in 23 unrelated Chinese patients☆,☆☆ Qiulan Ding a, 1, Wei Shen b, 1, Xu Ye c, Yingting Wu d, Xuefeng Wang a,⁎, Hongli Wang d a
Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Department of Laboratory Medicine, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Department of Hematology, Second affiliated Hospital of Guangzhou Medical University, Guangzhou, China d State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China b c
a r t i c l e
i n f o
Article history: Submitted 24 July 2012 Revised 8 August 2012 Available online 27 August 2012 (Communicated by U. Seligsohn, Ph.D., 9 August 2012) Keywords: Gene mutation Protein C deficiency Thromboembolism Thrombophilia
a b s t r a c t In this study, we investigated the clinical and genetic features of protein C deficiency in the Chinese population. A total of 23 symptomatic patients with protein C deficiency were identified by thrombophilic assays. Detailed clinical data about the patients with respect to their personal and family history of venous thromboembolism (VTE) were collected. Mutational analysis was then performed by direct sequencing of the protein C gene (PROC) in the patients and their family members. Of the 23 patients, 30.4% (7/23) had additional risk factors, 51.2% (12/23) suffered from recurrent thrombotic episodes, and 50.0% (6/12) of the patients with recurrent thrombosis had more than one heterozygous mutation in PROC itself or combined with protein S gene (PROS). The sex distribution of male:female was 19:4 in the 23 symptomatic patients and 10:2 in the 12 recurrent patients. Almost all patients (22/23) had lower extremity deep vein thrombosis (DVT) and one had pulmonary embolism (PE) only. A total of 15 different causative mutations were identified from the 23 subjects with 6 (40.0%) of the mutations being novel. Among the mutations identified, the Arg147Trp substitution was hotspot mutation in the Chinese population with a high frequency of 43.5%. Our finding suggests that complex genotypes of PROC or combined with protein S deficiency are primarily responsible for an increased risk of recurrent VTE. Our data further provides a framework for correlating the clinical pathogenesis of protein C deficiency to ethnic backgrounds in the Chinese population. © 2012 Elsevier Inc. All rights reserved.
Introduction Venous thromboembolism (VTE) is a common multifactorial disease resulting from the interaction of genetic and environmental risk factors. Genetic abnormalities of proteins involving the coagulation pathway that lead to hypercoagulability have been found in subjects suffering from thrombophilic disease. Factor V Leiden (FV Leiden) and prothrombin G20210A gene mutations are highly prevalent in the Caucasian population but are almost non-existent among the Asian population [1]. In contrast, a higher prevalence of protein Abbreviations: VTE, venous thromboembolism; DVT, deep venous thrombosis; PE, pulmonary embolism; PVT, portal venous thrombosis; MVT, mesenteric venous thrombosis; AVT, axillary vein thrombosis; PROC, protein C gene; PROS, protein S gene; PC:A, activity of protein C; PC:Ag, antigen of protein C. ☆ This study was partially supported by Shanghai Municipal Health Bureau of young scientific research project (2011Y195). ☆☆ Conflicts of interest and sources of funding: The authors stated that they had no interests which might be perceived as posing a conflict or bias. ⁎ Corresponding author at: Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, No. 197 Ruijin Second Road, Shanghai 200025, China. Fax: +86 21 64333548. E-mail address: [email protected] (X. Wang). 1 These authors made equal contributions to this work. 1079-9796/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bcmd.2012.08.004
S and protein C deficiency has been reported in the Asian population [2–5]. Two studies have reported that protein C and protein S deficiencies are the most important risk factors associated with thrombosis in Taiwanese and Japanese populations [3,4]. Protein C is a vitamin K-dependent serine protease zymogen synthesized in the liver. It is activated by thrombin in complex with thrombomodulin on the surface of endothelial cells. The activated protein C (APC), in complex with its cofactor protein S, inactivates procoagulant cofactors Va and VIIIa, thereby down-regulating thrombin generation in coagulation cascade. Protein C deficiency has an autosomal dominant pattern of inheritance and is associated with an increased risk of VTE. Heterozygosity for the disease has been shown to increase the risk of venous thrombosis by 5- to 10-fold [6]. Homozygosity and compound heterozygosity of protein C are associated with neonatal purpura fulminans or severe thromboembolic complications after birth. In rare cases, thrombotic episodes develop in childhood or adulthood. The prevalence of protein C deficiency defined by plasma levels is between 0.2% and 0.5% of the healthy population [7,8]. Most of the affected individuals remain asymptomatic throughout their life and 2–5% present clinically symptomatic protein C deficiencies, suggesting that protein C deficiency alone is a relatively low risk factor for thrombosis [9,10]. In Caucasians,
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the presence of the two common mutations, factor V Leiden and prothrombin G20210A, has been associated with a more severe phenotype in some protein C deficient patients [11,12]. However, available data are scarce regarding which kinds of additional genetic and environmental risk factors contribute to the onset of first-time and recurrent thrombotic episodes in patients with protein C deficiency in the Asian population. The human protein C gene (PROC) is localized in chromosome 2q13–q14 and comprises nine exons spanning more than 11 kb of genomic DNA. More than 300 mutations have been reported so far, and the spectrum of PROC mutations has been reported in several studies. Most reports are restricted to Western populations with only a few having been reported in the Asian population [11,13–15]. Results of these studies have indicated that the mutation profile of PROC is significantly influenced by ethnic background, thus, some mutations (Arg230Cys, Arg178Trp, Gln132X, Val297Met and Pro168Leu) are commonly found in the Caucasian population, while the others (Phe139Val, Arg169Trp, Val297Met, Met364Ile, and G8857del) are recurrent defects in the Japanese population. So far, few studies have been published on the spectrum of PROC defects and its thrombotic manifestation in patients with protein C deficiency in mainland China. In this study, we addressed this question by analyzing the molecular defects of the PROC in the 23 symptomatic subjects. Our results demonstrate a correlation between the ethnic background and the occurrence of thrombotic episodes stemming from protein C deficiency in the Chinese population. Materials and methods Patients This study was approved by the Ethics Committee of Ruijin Hospital, Shanghai Jiaotong University School of Medicine. Beginning in January 2002–January 2012, 28 unrelated patients with first-time or recurrent VTE episodes were referred to our thrombosis center due to their predisposition to the risk factors for VTE and finally were found that they were with protein C deficiency. All the index subjects were interviewed with respect to their medical history. The diagnosis of VTE and the presence of acquired risk factors, including immobilization, fractures, pregnancy, puerperium, oral contraceptives, hormone replacement, surgery, and malignancy, at the time of all episodes of VTE were recorded. The VTE diagnosis was based only on the results of objective investigations employing the following approaches: compression or color Doppler ultrasonography was used to diagnose deep venous thrombosis (DVT) and abdominal venous thrombosis; a high-probability ventilation–perfusion scan, pulmonary angiography, or computed tomography (CT) was used to diagnose pulmonary embolism (PE); magnetic resonance venography (MRV) or CT was used to diagnose occlusion of cerebral or abdominal veins. A detailed family history was obtained from the family members with special emphasis on the occurrence of prior VTE events. Hemostatic assays Venous blood samples from the patients and their family members were collected in 0.109 mmol/L sodium citrate after informed consent was obtained. Tests for thrombophilia including antithrombin (AT), protein C, protein S, plasminogen (PLG), tissue plasminogen activator (t-PA), lupus anticoagulant (LA), anticardiolipin antibody (ACA), anti-β2 glycoprotein I (anti-β2GPI), fibrinogen (Fg), and total homocysteine (Hcy). The plasma levels of ACA and anti-β2GPI (Euroimmun, Lübeck, Germany), and t-PA (Instrumentation Laboratory, Milan, Italy) were detected using enzyme-linked immunosorbent assays (ELISA) according to the manufacturers' instructions. LA was detected using a diluted viper venom time (DVVT) assay (Instrumentation Laboratory). The Hcy levels were determined using the AxSYM homocysteine kit
(Abbott, Lake County, IL, USA) based on a fluorescence polarization immunoassay (FPIA). The functional and antigenic fibrinogen levels were detected using the Clauss method on a Sysmex CA7000 analyzer (Sysmex Corporation, Tokyo, Japan) and immunoturbidimetric assay, respectively. The activities of protein C, AT, and PLG (PC:A, AT:A, and PLG:A) were analyzed using chromogenic substrate methods (Instrumentation Laboratory). The activity of free protein S (FPS:A) was assayed using a clotting method based on prothrombin time (Instrumentation Laboratory). Free protein S antigen (FPS:Ag) was measured with a polyclonal anti-human protein S antibody using the ZYMUTEST free protein S kit (Hyphen BioMed, Neuville-Sur-Oise, France). Protein C antigen (PC:Ag) was determined using a sandwich ELISA method with a rabbit anti-human protein C polyclonal antibody (Dako, Glostrup, Denmark) as a capture antibody and a horseradish peroxidase (HRP) conjugated antibody as a detection antibody. Protein C deficiency was diagnosed only in cases where the activity of protein C (PC:A) was lower than the normal level and both liver and renal functions were normal. Genetic analysis of PROC Genomic DNA was extracted from the peripheral blood leukocytes using the QIAamp DNA mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Mutations in the PROC and protein S gene (PROS) were identified as previously described [5]. The detected mutations were confirmed by reverse sequencing and were verified using a second amplicon. To rule out polymorphisms, the novel missense mutations were screened in 50 normal individuals. Only the corresponding sequence was amplified and sequenced in family members. The genetic alterations were reported according to both the standard international nomenclature guidelines recommended by the Human Genome Variation Society (HGVS; http://www.hgvs.org/mutnomen/recs.html), with nucleotide + 1 as the A of the ATG translation initiation codon and Foster's numbering system. The genomic DNA (GenBank: NM_000312.2) and cDNA (GenBank: P04070) sequences of PROC were used as references. Results Association between the genetic variants of PROC and onset of thrombotic episodes A total of 28 patients were diagnosed as having protein C deficiency, 2 patients had combined protein C and protein S deficiency, defined by the levels of PC:A and PS:A in their plasma. Genetic defects in the PROC were identified in 23 unrelated patients. No PROC mutation was identified in the other five patients, who were diagnosed with acquired protein C deficiency due to liver diseases. One heterozygous mutation in the PROS was found in one of the two patients with combined protein C and protein S deficiency, but no mutation was identified in the other patient with an FPS:A level of 18.0% and a FPS:Ag level of 23.7%, respectively. The subject's mother had a similarly low FPS level, suggesting that the protein S deficiency may be hereditary in the family. The characteristics of the genetic defects and the onset of thrombotic episodes in the 23 unrelated symptomatic patients with protein C deficiency are shown in Table 1. Of the 23 symptomatic subjects, 30.4% (7/23) had additional risk factors. Of the seven patients, four presented the acquired risk factors at the time of onset of VTE: surgery in patient 7 and patient 15; puerperium in patient 13; and the thrombotic complication related to the use of a permanent filter and/or advanced age in patient 10. Three patients exhibited other genetic risk factors: an elevated homocyteine level in patient 21, protein C and protein S deficiency coexisted in patients 5 and 6. Results of all the other thrombophilic assays were normal in the 23 symptomatic patients (data not shown). Twelve patients (51.2%) suffered from recurrent thrombotic episodes, six of
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55
Table 1 Characteristics of the genetic defects and onset of thrombotic episodes in 23 unrelated Chinese patients with protein C deficiency. No. of patients
Age/ sex
Activity (%)
Antigen (%)
Nucleotide variation (DNA)
Amino acid substitution†
1
16/M
21.0
20.0
g.26T>C g.8599C>T
2
20/M
19.0
42.0
3
28/M
21.0
18.4
4
27/M
39.0
42.1
5
30/M
36.0 18.0a 57.0 23.7.b
6
24/M
46.0 19.5a 64.4 20.9b
7
47/M
30.0
48.4
8
7/M
26.0
46.1
p.Leu8(−34)Pro p.Thr337 (295) Ile g.1474G>A p.Glu71 (29) Lys g.6152C>T p.Arg189 (147) Trp p.Arg220 (178) g.6245C>T g.8478G>C Trp p.Asp297 (255) His g.5498C>T p.Arg57 (15) Trp g.6152C>T p.Arg189 (147) Trp p.Leu8 (−34) g.26T>C Pro# g.7212 C>T g.6161-6163delAAG p.Ala251 (209) Val# p.Lys192 (150) del g.6152C>T p.Arg189 (147) c.1680T>Ac Trp p.Tyr560X (Tyr519X)d g.6152C>T p.Arg189 (147) g.6245C>T Trp# p.Arg220(178) Trp# g.5498 C>T p.Arg57(15)Trp
9
37/M
64.0
80.9
g.3426G>T
10
84/F
54.6
11
24/F
65.9
76.5
12
24/F
38.6
45.3
13
33/F
63.9
71.8
14
19/M
69.4
74.9
15
68/M
46.8
67.2
16
28/M
63.9
64
17
28/M
68.7
68.2
18
24/M
55.0
ND
19
64/M
65.0
ND
20
69/M
45.6
71.0
21
23/M
65.3
75.6
22
42/M
63.0
ND
23
40/M
49.0
43.2
p.Asp170(128) Tyr g.3442G>A p.Cys175(133) Tyr g.6152C>T p.Arg189(147) Trp g.6152C>T p.Arg189(147) Trp g.6152C>T p.Arg189(147) Trp g.6152C>T p.Arg189(147) Trp g.6152C>T p.Arg189(147) Trp g.6152C>T p.Arg189(147) Trp g.6161-6163delAAG p.Lys192(150) del g.8478G>C p.Asp297(255) His g.8478G>C p.Asp297(255) His g.8746T>C p.Leu386(344) Pro g.8763G>A p.Gly392(350) Arg g.8807G>A p.Met406(364) Ile g.8831G>A p.Trp414(372)X
Thrombotic episodes (onset age)
Thrombophilic factors
Thrombotic episodes (family member)
DVT (14), DVT (15)
PE (19)
DVT, PVT (father with p.Glu29Lys)
DVT(21), MVT(21), DVT (23)
DVT (mother with p.Asp255His)
DVT(25), DVT(26)
DVT (28y), DVT (30)
Protein S deficiency
DVT (mother with p.Lys150del combined with protein S deficiency), MVT (brother with the double mutations)
PE (22), DVT(22), DVT (24)
Protein S deficiency
DVT (father with p.Tyr519X in PROS)
DVT (40y), DVT (43)
Cholecystectomy
DVT (brother with the double mutations)
DVT(4), DVT(7), AVT (7) DVT (37) DVT(79), DVT(83)
Permanent filter, advanced age
DVT (21) DVT (23), DVT (24) DVT (33)
Puerperium
DVT (19) Both DVT and PE (68)
Cholecystectomy
DVT (28) DVT (27) DVT (23) DVT (62), DVT (63) DVT (69) DVT (23)
Hyperhomocysteinemia
DVT (37), DVT (41)
DVT (mother with p.Met364Ile)
DVT (39), DVT (40)
DVT (sister with p.Trp372X)
Reference range: PC:A: 70–140%; PC:Ag: 70–130%; FPS:A: 63–135%; FPS: Ag: 60–150%. † : The numbering of changed amino acid residue was given according to the beginning from the mature protein C or protein S within parentheses; #: double mutations. DVT: deep venous thrombosis (indicating the low extremity here); PE: pulmonary embolism; PVT: portal venous thrombosis; MVT: mesenteric venous thrombosis; AVT: axillary vein thrombosis. ND: no detection. a FPS:A. b FPS: Ag. c PS cDNA. d PS amino acid substitution.
these patients (patient 1, patients 3–7) had more than one heterozygous mutation in PROC and PROS: patient 1 and patients 3–4 carried compound heterozygous PROC mutations with the ranges of protein C levels in activity from 21.0% to 39.0% and antigen from 18.4% to 42.1%; patient 5 had a PC:A of 36.0% and a PC:Ag of 57.0%, a FPS:A
of 18.0% and a FPS:Ag of 23.7%, this patient also carried triple heterozygous PROC variants, but no genetic defect was found in the PROS. A heterozygous mutation was identified in both the PROC and PROS in patient 6. Patient 7 may exhibit two mutations on the same allele, predicted by his brother, who inherited the same mutations, and his
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Table 2 Genetic defects in the PROC identified in 23 unrelated Chinese patients with protein C deficiency. Mutations
No. of families
Leu8 (−34) Pro Arg57 (15) Trp Glu71 (29) Lys Asp170 (128) Tyr Cys175 (133) Tyr Arg189 (147) Trp Arg220 (178) Trp Ala251 (209) Val Asp297 (255) His Thr337 (295) Ile Leu386 (344) Pro Gly392 (350) Arg Met406 (364) Ile Trp414 (372) X Lys192 (150) del Total
2 2 1 1 1 10 2 1 3 1 1 1 1 1 2
Reported in Asia
Reported in worldwide
Reported in this study
+ + + + +
Other reported mutations in the same residue Arg15Gln, Gly Glu29X
+ + +
Arg178Gln,Pro
+ + + + + + + 5
4
Trp372Arg
6
The numbering of changed amino acid residue was given according to Foster's numbering (beginning from the mature protein C) within parentheses.
sister, who had normal PROC. Only one heterozygous PROC mutation was identified in each of the remaining six subjects (patients 8, 10, 12, 19, 22 and 23) with recurrent VTE events. Patient 8, with low levels of PC:A and PC:Ag (26.0% and 46.1%, respectively), developed recurrent DVT at a very early age. Recurrent DVT was associated with advanced age in two subjects (patients 10 and 15). No predisposing risk factors were detected in the other three patients other than the heterozygous protein C deficiency. At least one family member had VTE in 7 of the 23 unrelated families; 6 of these occurred in the pedigrees with patients having recurrent DVT. Considering the localization of the thromboembolic events, all of the other 22 subjects (95.7%) had lower-extremity DVT except for patient 2, who had compound heterozygous mutations in PROC and manifested only PE. Of the 22 subjects, patients 6 and 15 also had PE; patient 3 had both DVT and mesenteric venous thrombosis (MVT); and patient 8 had an axillary vein thrombosis (AVT) together with DVT. The mean age at the time of the first thrombotic episode was 33.1 years (range: 4 to 79 years) in the 23 symptomatic patients. In patients 1–7 who had more than one variant in the PROC and PROS, the average age of the first episode of VTE was 24.1 years. Four subjects (patients 1, 2, 8 and 14) experienced VTE before the age of 20. Sex distribution of male:female was 19:4 in the 23 symptomatic patients and 10:2 in the 12 recurrent patients, respectively, suggesting that of those with protein C deficiency, male may have a much higher frequency of developing first and recurrent VTE than does female in Chinese population.
Characteristics of genetic defects of PROC in the 23 unrelated patients In this study, we reported the spectrum of the PROC mutations in 23 subjects with protein C deficiency. A total of 15 different causative mutations were identified including 13 missense mutations (86.7%), one nonsense mutation (6.7%), and one in-frame deletion (6.7%). Among the 15 identified mutations, 6 (40.0%) had not been mentioned either in the database or in the literature; 4 (26.7%) had been reported worldwide and 5 (33.3%) only in Asia. Of the 6 novel mutations, 5 were missense mutations, including Glu29Lys in the gamma-carboxyglutamic (Gla) domain, Asp128Tyr and Cys133 Tyr in the epidermal growth factor (EGF)-like domain, Thr295Ile, Thr315Ala, Leu344Pro in the serine protease domain, and the last one was a nonsense mutation (Trp372X) located in the serine protease domain (Table 2).
While six mutations were found in more than one patient, the Arg147Trp mutation occurred 10 times with a frequency of 43.5% (10/23) and the Asp255His occurred three times (13.0%). All remaining four mutations (Leu-34Pro, Arg15Trp, Lys150del and Arg178Trp) occurred two times. The high recurrence incidence of the Arg147Trp mutation suggested but did not prove a founder effect in the Chinese population (Table 2). Discussion This is the first report that describes both the clinical and genetic features of protein C deficiency in 23 unrelated subjects in the Chinese population. It also outlines the relationship among the genetic alterations of PROC with respect to the onset of thrombotic episodes. Heterozygous protein C deficiency is inherited in an autosomal dominant fashion, and its frequency in the Chinese population is approximately 0.29% [5]. Several studies have suggested that additional genetic risk factors can coexist and contribute to thrombotic risk in patients with protein C deficiency. Due to a high prevalence of FV Leiden in a general Caucasian population (prevalence, 2–7%), and in patients (prevalence, 20–40%) with thrombosis [16], the combination of protein C deficiency plus FV Leiden is relatively common. A previous report found that 14% of a group of 113 unrelated protein C heterozygotes also had the FV Leiden [11]. In another report, 73% of family members who were heterozygous for both protein C deficiency and FV Leiden mutation manifested thrombosis. In contrast, 31% and 13% of family members having either the protein C deficiency or FV Leiden mutation, respectively, had experienced a thrombotic episode [12], indicating that protein C deficiency combined with FV Leiden mutation presents an increased risk of thrombosis. However, few cases of FV Leiden mutation have been identified in either Asian or African populations. In this study, of the 23 patients with symptomatic protein C deficiency, 30.4% (7/23) had additional risk factors. Four patients presented acquired risk factors, and three patients exhibited other genetic risk factors. These results are consistent with the previous report that the initial episode of VTE in patients with protein C deficiency is associated with other genetic and acquired risk factors in approximately 30% of the subjects [17]. Combined protein C and protein S defects are rare and have been reported in association with ischemic strokes, multiple aortic thrombosis, mesenteric artery thrombosis, DVT, intracardiac multichamber thrombi, avascular necrosis, and portal vein thrombosis (PVT) [18–21]. The prevalence of thrombotic events among patients with combined protein C and
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protein S defect has not been reported. Here we have identified two subjects with combined protein C and protein S deficiency, who had their first thrombotic events in their early twenties. In addition, at least one family member suffered from VTE in each of the two pedigrees. We predict that, unlike in Caucasians, coexisting protein S deficiency rather than FV Leiden mutation may contribute to a more severe risk of thrombosis in subjects with protein C deficiency in the Chinese population. Surprisingly, in one case identified among the 3493 Chinese adult blood donors and healthy individuals, a 40-year-old male with coexisting protein C and protein S deficiency had never experienced thrombotic events nor had his family members, indicating that an asymptomatic carrier of both heterozygous protein C and protein S deficiency can exist as a normal individual [5]. Severe homozygous or compound heterozygous protein C deficiency occurs in approximately 1 in 500,000 to 1 in 750,000 live births. Homozygous and compound heterozygous protein C deficiencies are classically associated with neonatal purpura fulminans (NPF), massive thrombosis, and disseminated intravascular coagulation (DIC) at birth. Occasionally, patients with low levels of PC:A present with VTE in childhood or adolescence. In this study, we found a very high frequency of complex genotypes of PROC in the 23 symptomatic subjects and in the 12 recurrent VTE subjects, including one double heterozygous, four compound heterozygous, and one triple heterozygous mutations. Of these, five had recurrent VTE events and one subject experienced only a PE episode. One of the possible reasons may be that most patients in our study were recruited consecutively among outpatients who had idiopathic first and recurrent episodes of VTE at a young age, without definite acquired risk factors. Therefore, the onset of VTE tends to be provoked by inherited thrombophilic defects. Thus, we predict that complex genotypes of PROC may play an important role in the protein C deficient patients with recurrent VTE in the Chinese population. In our report, 95.7% of symptomatic protein C deficient subjects (22/23) had thrombosis in the deep veins of the lower extremities and one only had PE. Co-occurrence of thrombotic events was recorded as well: PE in patients 6 and 15 (9.1%), MVT in patient 3 (4.5%), and AVT in patient 8 (4.5%). The much higher frequency of lower extremity DVT was consistent with previous reports that the co-occurrence of other types of VTE is low in patients with protein C deficiency [22,23]. Of the 23 symptomatic patients, the average age at onset of the first thrombotic episode was 33.1 years, which is consistent with a previous study which demonstrated that in heterozygous individuals thrombotic episodes occur at age 30–40 years and is rare before the age of 20 years [22]. Whereas in the six patients with complex genotypes of PROC (patient 1–5, 7), the average age of the first onset of VTE was earlier (24.5 years). As expected for an autosomal genetic disorder, the prevalence of hereditary protein C deficiency is similar between males and females. However, there is firm evidence that the male sex in itself is an important risk factor for VTE. Males not only have a higher risk of a first VTE (1.4-fold) than females, but they also have a 2.5-fold higher risk of thrombotic recurrence than do females [24,25]. Moreover, the idiopathic first thrombotic events were more common in males, while first thrombotic events associated with special risk factors (e.g., oral contraceptive use, pregnancy, the postpartum state, and estrogen-containing hormonal therapy) were reported almost five times more often in women. We found a high male:female ratio in the symptomatic subjects (19:4) and recurrent subjects (10:2) but a low ratio in the 10 healthy subjects (3:7) [5], revealing that male patients with protein C deficiency have a much higher frequency of venous thromboembolic events than do females. A clear explanation for this finding is unknown and further investigation of the interaction between the sex of the subject and protein C deficiency contributing to VTE may need to be conducted. In China, only 2.22% of women of childbearing age take an oral contraceptive, whereas most of them choose an intrauterine device (49.13%) or surgical sterilization (36.25%) for birth control, according to the
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statistics of the State Family Planning Commission in 2009. This may reduce the oral contraceptive use associated with VTE occurrence in the individuals with protein C deficiency in the Chinese population. We identified a total of 15 different causative mutations of PROC in the 23 subjects with protein C deficiency, including 13 missense mutations (86.7%), one nonsense mutation (6.7%), and one in-frame deletion (6.7%). Of these, 40.0% (6/15) were novel mutations, indicating that the mutation spectrum of PROC is heterogeneous in the Chinese population. In contrast, we found one ethnic-specific mutation: the Arg147Trp mutation which occurred 10 times (43.5%) in 23 unrelated subjects (Table 2). The Arg147Trp mutation was first identified in an American of Hawaiian descent [26], and the high prevalence of heterozygotes (0.85%) for this mutation has been reported for the normal Chinese population. In addition, Arg147Trp was also the most common genetic defect among symptomatic patients in the Taiwanese population, and more than half of the symptomatic patients had the mutation in combination with other protein C or protein S deficiency and/or environmentally precipitating factors [27]. Similar results were found in our study: 60% of patients (6/10) had a similar combination of risk factors to those reported in the previous study, indicating that Arg147Trp mutation is associated with VTE but is a relatively weak risk factor. To sum up our previously reported mutations of PROC in 13 unrelated subjects [5,28,29], we found two significant ethnicity-specific mutations: Arg147Trp and Lys150del occurred 10 times (27.8%) and 5 times (13.9%), respectively, in the 36 unrelated subjects. The Lys150del was first reported in the Japanese population [13], and a subsequent study showed that it was one of the most prevalent mutations in the Japanese population [3]. Considering their high frequencies, further biochemical and functional analysis of these two hotspot protein C mutations (Arg147Trp and Lys150del) in the Chinese population is worth pursuing. In conclusion, we have demonstrated that the clinical and genetic features of protein C deficiency have some unique differences among Chinese, Caucasian, and perhaps even other Asian populations. Unlike the Caucasian population, complex genotypes of PROC or combination of protein C and protein S deficiency are mainly responsible for an increased risk of VTE, especially for recurrent VTE. For the first time, we have demonstrated that male patients with protein C deficiency have a much higher frequency of VTE than do females in the Chinese population. Our results may explain the molecular basis for the pathogenesis of protein C deficiency according to ethnic background and may help to construct pathogenesis-based prophylaxis and treatment strategies for VTE in the Chinese population. Acknowledgments The authors wish to thank Audrey Rezaie for proofreading the manuscript and thank patients and their family members for their participation in this study. References [1] M. Margaglione, E. Grandone, Population genetics of venous thromboembolism. A narrative review, Thromb. Haemost. 105 (2011) 221–231. [2] P. Angchaisuksiri, Venous thromboembolism in Asia—an unrecognised and under-treated problem? Thromb. Haemost. 106 (2011) 585–590. [3] T. Miyata, Y. Sato, J. Ishikawa, et al., Prevalence of genetic mutations in protein S, protein C and antithrombin genes in Japanese patients with deep vein thrombosis, Thromb. Res. 124 (2009) 14–18. [4] M.C. Shen, J.S. Lin, W. Tsay, Protein C and protein S deficiencies are the most important risk factors associated with thrombosis in Chinese venous thrombophilic patients in Taiwan, Thromb. Res. 99 (2000) 447–452. [5] T. Zhu, Q. Ding, X. Bai, et al., Normal ranges and genetic variants of antithrombin, protein C and protein S in the general Chinese population. Results of the Chinese hemostasis investigation on natural anticoagulants study I group, Haematologica 96 (2011) 1033–1040. [6] B. Dahlback, The protein C anticoagulant system: inherited defects as basis for venous thrombosis, Thromb. Res. 77 (1995) 1–43. [7] J. Miletich, L. Sherman, G. Broze Jr., Absence of thrombosis in subjects with heterozygous protein C deficiency, N. Engl. J. Med. 317 (1987) 991–996.
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