Yu Hu, Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430022, China. Tel.: +86 27 85726335; fax: +86 27 85776343. E-mail: firstname.lastname@example.org
See also Hamasaki N. Unmasking Asian thrombophilia: is APC dysfunction the real culprit? This issue, pp 2016–8.
Summary. Background: There are ethnic differences in the genetic risk factors for venous thrombosis (VT). The genetic causes of VT in the Chinese population are not fully understood.
Objectives: To identify possible common abnormal factors that could contribute to thrombosis susceptibility.
Methods/Results: We measured the levels of nine types of plasma coagulation factor, three types of anticoagulation factor and two types of fibrinolytic factor in 310 VT patients. Factor V activity was higher in 32 cases. Eleven of the 32 cases also had low protein C (PC) or protein S (PS) activities, indicating PC or PS deficiency. No other abnormalities were observed in the other 21 cases. All of the samples were sensitive to activated PC inactivation. Therefore, the abnormal factor involved may be FV inactivator or its cofactor rather than FV itself. Resequencing identified a common PROC c.574_576del variant in 10 of the 32 subjects. In a case–control study, this variant was detected in 68 of the 1003 patients and in 25 of the 1031 controls. It had an adjusted odds ratio of 2.71 (95% confidence interval [CI] 1.68–4.36). PC amidolytic activities of most variant carriers were similar to those of non-carriers, but the mean anticoagulant activity was only 72.7 U dL−1. Expression studies in vitro showed that the anticoagulant activity of the mutant PC was 43.6% of that of the wild-type PC.
Conclusions: We identified what is, so far, the most common genetic risk factor for VT in the Chinese population, with its prevalence being approximately 2.36%.
Venous thrombosis (VT) is one of the leading causes of morbidity and mortality throughout the world. Because of a paucity of epidemiologic data from non-Western countries, it has long been considered to be a disease that is only prevalent in white populations. Recent studies showed that VT is as prevalent, if not more so, in the black and Asian populations [1–4]. Major hazards of VT include a disabling post-thrombotic syndrome and sudden death resulting from pulmonary embolism [5,6]. It is believed that both genetic and acquired risk factors are implicated in VT [7–10]. Family and twin studies have indicated that genetic factors make a contribution of ∼ 60% to VT [11,12]. Therefore, definition of the genetic architecture of VT may help in risk prediction, secondary prevention , and the development of novel therapies.
Three common variants, factor V Leiden (F5 R506Q), prothrombin gene G20210A, and antithrombin Cambridge II, which are known to be associated with an increased risk of VT in Caucasians [14–16], are rare in other populations, including Asians. It has been concluded that Asian thrombophilia is a protein C (PC) system dysfunction caused by various rare mutations, on the basis of the results from previous studies from Taiwan, Hong Kong, Japan, and Thailand [17–22]. However, these rare mutations can still explain only a fraction of the VT events. Therefore, we set out to determine whether there were any other prevalent genetic variants associated with VT in the Chinese population. As most of the known risk factors for VT are associated with blood stasis, a blood hypercoagulable state, a blood hypoanticoagulable state, or hypoactive fibrinolysis [23–26], we mainly focused on plasma factors that are involved in blood coagulation in the screening stage.
Materials and methods
Subjects and sample collection
Sample and data collection and ascertainment of VT events in the case–control study have been described previously . Unselected consecutive VT patients (n = 1003) were recruited between 1 March 2008 and 30 June 2011 at the Hubei Clinical and Research Center of Thrombosis and Hemostasis. The criteria for diagnosis of VT are described in the guidelines for diagnosis and treatment of VT, China, 2008 , which are the same as the global guidelines. Briefly, validation of VT incidents (deep vein thrombosis [DVT] and pulmonary thromboembolism) were based on clinical manifestations, D-dimer assay, and objective techniques (compression ultrasound or venography for DVT, and ventilation/perfusion lung scanning, computed tomography angiography or pulmonary angiography for pulmonary thromboembolism). Age-matched and sex-matched control participants from central China (n = 1031) and eastern China (Shanghai, n = 492), without an individual or family history of thrombosis (arterial thrombosis such as myocardial infarction and VT), were enrolled during the same period. The case group and control group had a similar median age (52 years vs. 51 years), mean age (51.5 ± 14.4 years vs. 50.3 ± 14.5 years), 5th–95th percentile age (25.0–74.0 years vs. 25.6–73.0 years), and sex distribution (53.0% male). This study was approved by the ethics committee of Union Hospital, Huazhong University of Science and Technology, and complied with the principles expressed in the Declaration of Helsinki. Written informed consent was obtained from all participants.
Coagulation screening tests
Plasma samples from 310 patients were randomly selected for abnormal factor screening. Procoagulant activities of FII, FV, FVII, FVIII, FIX, FX, FXI, FXII and activated PC (APC) cofactor activity of protein S (PS) were determined with a clotting assay. Antithrombin activity, PC amidolytic activity, plasminogen level and plasminogen activator inhibitor-1 (PAI-1) level were measured with a chromogenic method. Fibrinogen level was measured using the Clauss assay. All of the kits and reagents for these laboratory tests were purchased from Dade Behring-Siemens Healthcare Diagnostics (Marburg, Germany). The APC sensitivity of FV was determined with a modified APC resistance assay (Chromogenix, Milan, Italy). Tests were carried out on an automated Sysmex CA 7000 Analyzer (Sysmex, Kobe, Japan), according to the manufacturer’s instructions. The normal ranges were established in 78 healthy controls. These reference intervals, in terms of mean ± standard deviation (SD), were as follows: 3.0 ± 0.5 g L−1 for fibrinogen level, 97 ± 16 U dL−1 for PC amidolytic activity, 103 ± 18 U dL−1 for PS cofactor activity, 105 ± 11 U dL−1 for FII activity, 113 ± 13 U dL−1 for FV activity, 103 ± 14 U dL−1 for FVII activity, 108 ± 22 U dL−1 for FVIII activity, 103 ± 16 U dL−1 for FIX activity, 110 ± 14 U dL−1 for FX activity, 103 ± 11 U dL−1 for FXI activity, 102 ± 14 U dL−1 for FXII activity, 106 ± 14 U dL−1 for plasminogen activity, and 2.73 ± 1.40 U mL−1 for PAI-1 activity.
Genetic resequencing and variant identification
All of the exons, intron–exon boundaries and 5′/3′-untranslated regions of the PROC gene (encoding PC) and the PROS1 gene (encoding PS) were amplified and then resequenced in subjects with evidently higher FV activity in the coagulation screening stage. Primer sequences and annealing temperatures used in PCR are summarized in Table S1. Sequence variants and amino acid changes were designated according to current nomenclature [29,30]. The accession numbers were NM_000312.3 and NM_000313.3, respectively.
The identified variant, PROC c.574_576del, was subsequently detected in all of the cases and controls by employing a PCR–restriction fragment length polymorphism (PCR-RFLP) method (Fig. 1). This variant allele abolishes a recognition site for MboII. The primer sequences for amplification were 5′-GGAGTGGTCTAAGTATCATTGGTTC-3′ and 5′-TTGGTCTTCTTGGTATTCTGTGTC-3′. Digestion was carried out by incubating 8.5 μL of the PCR product in 1 μL of 10 × buffer B with 0.5 U of MboII (Fermentas, Burlington, Canada) for 2 h at 37 °C. The digestion products were separated by 2% agarose gel electrophoresis. Altogether, 96 DNA samples were selected and subjected to sequencing to verify the genotyping results determined with the PCR-RFLP assays. The genotyping data from the PCR-RFLP assays were completely consistent with those obtained by sequencing.
Association between PROC c.574_576dup/del genotype and PC activity
To determine whether this variant affects PC activity, we compared the mean plasma PC activity of variant carriers (heterozygotes) with that of non-carriers. Subjects with severe PC deficiency caused by rare mutations were excluded. Finally, plasma samples from 69 variant carriers (50 patients and 19 controls) and 72 age-matched and sex-matched non-carriers (50 patients and 22 controls) were investigated for amidolytic activity and anticoagulant activity of PC with a chromogenic method and a clotting assay, respectively. PC anticoagulant activity was determined with the HEMOCLOT Protein C kit (Hyphen BioMed, Andresy, France). Two polymorphisms (rs1799808 and rs1799809) in the promoter region of the PROC gene were genotyped by PCR sequencing. Multiple linear regression analysis was used to evaluate the association between the PROC gene single-nucleotide polymorphisms (SNPs) and PC anticoagulant activity levels in these 141 subjects.
In vitro expression studies
To produce the PROC c.574_576del mutant, mutagenesis was performed by high-fidelity PCR with the wild-type human PC open reading frame expression-ready clone (GeneCopoeia, Rockville, MD, USA) as template. The mutant construct was confirmed by sequencing. COS-7 cells (Cellbank of the Chinese Academy of Sciences) and HEK-293 cells (a gift from X. Gao, originally purchased from ATCC) were grown in Dulbecco’s modified Eagle’s medium (Gibco-BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum, 2 mm glutamine, 100 U mL−1 penicillin, and 100 μg mL−1 streptomycin, and maintained at 37 °C in 5% CO2. Approximately 90% confluent cells in 35-mm-diameter six-well plates were cotransfected with 3.2 μg of mock vector, wild type, or mutant PC constructs, and 0.8 μg of the enhanced green fluorescent protein (EGFP) control vector with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The EGFP control vector was used to standardize the transfection efficiency. After 12 h of transfection, the medium was changed to serum-free medium supplemented with 10 mg L−1 vitamin K1, and harvested 72 h later. Quantification of the mutant transcription level as compared with the wild-type level was performed by quantitative PCR on an ABI StepOne Plus real-time fluorescence quantitation instrument with Platinum SYBR Green qPCR SuperMix (Invitrogen), according to the manufacturer’s instructions. The glyceraldehyde-3-phosphate dehydrogenase gene was used as a reference. Primer sequences for mutagenesis and quantitative PCR are summarized in Table S1. The antigen concentration of the wild-type and mutant PC from culture media of transfected cells was measured by the ELISA method with the ZYMUTEST Protein C kit (Hyphen BioMed). Stable transformants were further established with G-418 (neomycin) to determine the functional activity of recombinant PC, as previously described [31,32]. The serum-free conditioned media of stable transfected HEK-293 cells were filtered with a Vivacon 2 Centrifugal Ultrafiltration device (Sartorius, Goettingen, Germany), and were then diluted in 10 mm Tris-HCl and 150 mm NaCl (pH 7.4) to provide a range of final PC antigen level of 0–100 U dL−1. It is believed that recombinant vitamin K-dependent proteins expressed from mammalian cells are not fully γ-carboxylated, and should be properly purified. Nevertheless, we utilized the centrifugation step instead of carboxylated PC isolation, assuming that the efficiencies of γ-carboxylation in the wild-type and mutant PC media would be similar under the same conditions [33,34]. Then, the wild-type and mutant PC activity were compared by use of both the chromogenic method and the clotting method.
For measurement data, differences between groups were analyzed with a Student’s t-test or non-parametric tests. A chi-squared test was used for enumeration data. Deviations from Hardy–Weinberg expectations were assessed with a chi-squared test. Statistical power was estimated with the Power and Sample size calculation program . Multivariate logistic regression analysis was carried out to test whether a significant association between the identified gene variant and VT persisted after adjustment for selected confounders (age, gender, smoking status, malignant tumor, pregnancy/puerperium, sedentariness/immobilization, and the PROC c.565C>T mutation). Regression analysis was used to evaluate the association between the PROC gene SNPs and the anticoagulant activity level of PC. A two-tailed P-value of <0.05 was considered to be statistically significant. All analyses were carried out with spss version 12.0 (SPSS, Chicago, IL, USA).
FV procoagulant activities were significantly higher (> 152 U dL−1, mean + 2 SD) in 32 cases. Eleven of the 32 cases also had low PC or PS activities, indicating PC or PS deficiency. However, no other abnormalities were observed in the other 21 cases (Fig. S1). Modified APC resistance tests showed that all 32 subjects had FVa that was sensitive to APC inactivation (mean APC-sensitive ratio, 3.04 ± 0.15). These data suggested that the abnormal factor might be FV inactivator (PC) or its cofactor (PS) rather than FV itself, and that the possible abnormality might not be easily detected by traditional tests.
Genetic resequencing and variant identification
Some rare mutations and neutral polymorphisms were identified in the 11 individuals with higher FV activity and low PC or PS activity. These genetic features are shown in Table S2. Moreover, a PROC c.574_576del variant was identified in 10 patients with higher FV activity but normal PC and PS activity. In an extended analysis of 100 controls, this heterozygous variant was also present in two subjects, indicating that this variant was a common polymorphism in the study population.
The PROC variant and VT risk
As shown in Table 1, the PROC c.574_576del variant was identified in 68 (6.78%) of the cases and in 25 (2.42%) of the controls. Only one PROC c.574_576del homozygote was identified, in the case group. In controls, the homozygous variant was absent. This variant was also detected and confirmed in 11 of the 492 (2.24%) healthy participants from eastern China, indicating that this variant is likely to be common in populations from other regions of China as well. In addition, the PROC c.565C>T mutation in the heterozygous state was present in 59 (5.88%) of the patients and in nine (0.87%) of the controls, as previously described . No homozygote for this mutation was identified. The crude odds ratios (ORs) were 2.93 (95% confidence interval [CI] 1.84–4.67, for the PROC c.574_576del variant) and 7.10 (95% CI 3.50–14.39, for the PROC c.565C>T mutation), as compared with individuals without the two PC variants. The statistical power for the PROC c.574_576del variant was approximately 0.996, with a significance level of 0.05. Multivariate logistical regression analysis revealed that the association between the two PROC gene variants and VT was still significant, with ORs of 2.71 (95% CI 1.68–4.36, for the PROC c.574_576del variant) and 7.13 (95% CI 3.49–14.56, for the PROC c.565C>T variant), after correction for other risk factors in a dominant model.
Table 1. Association between the PROC c.574_576del variant and venous thrombosis in the Chinese population
Cases, no. (%)
Controls, no. (%)
OR (95% CI)
OR (95% CI)
CI, confidence interval; OR, odds ratio; VAF, variant allele frequency; dup, the PROC c.574_576dup common allele; del, the PROC c.574_576del variant allele. The genotyping data obtained from patients and controls were used to calculate the ORs of the PC variants for venous thrombosis. Test for Hardy–Weinberg equilibrium of the c.574_576dup/del polymorphism: P =0.694. *Data were calculated by unconditional logistic regression adjusted for age, gender, smoking status, malignant tumor, sedentariness/immobilization, pregnancy/puerperium, and the PROC c.565C>T mutation.
3.31 × 10−10
6.88 × 10−8
3.80 × 10−6
4.04 × 10−5
dup/del + del/del
2.59 × 10−6
4.59 × 10−5
2.28 × 10−6
In particular, three patients were double heterozygous carriers of both the PROC c.565C>T and the PROC c.574_576del variants on different alleles (confirmed by clone sequencing), and no controls bearing this genotype were identified. All three double heterozygous individuals experienced only one VT event, at the ages of 32, 39 and 53 years, respectively. Their mean PC amidolytic activity was 47.65 ± 3.79 U dL−1, slightly lower than that of PROC c.565C>T carriers (51.73 ± 6.92 U dL−1). However, the difference was not statistically significant (P =0.34). Because of the rarity of this double heterozygous genetic trait in both controls (n = 0) and cases (n = 3), we could not assess the relative risk of developing VT in double heterozygotes. A large thrombophilic family cohort study should be carried out in the future.
Carriers of the two PC variants on the same allele were not observed in the whole study population that we enrolled.
Association between the PROC c.574_576dup/del genotype and PC activity
As shown in Fig. 2, the mean PC amidolytic activity levels of normal individuals and variant carriers were 99.5 ± 13.4 U dL−1 and 95.0 ± 11.0 U dL−1, respectively. Although the difference was statistically significant (P =0.045), it was minor. However, the mean PC anticoagulant activity of variant carriers was 72.7 ± 9.1 U dL−1, much lower than that of non-carriers (101.3 ± 11.9 U dL−1, P =1.02 × 10−22). For each variant carrier, PC anticoagulant activity as measured with the clotting assay was lower than amidolytic activity measured with the chromogenic method. However, the PC anticoagulant activities of several heterozygous individuals were greater than those of several normal individuals, indicating that other genetic or environmental factors might affect PC activity.
Linear regression (Table 2) showed that the PROC c.574_576dup/del variant had the most significant correlation with the anticoagulant activity level of PC, with a regression coefficient of − 0.763. Table 3 shows mean PC anticoagulant levels for various combinations of the three polymorphisms.
Table 2. Association between the PROC gene polymorphisms and protein C (PC) anticoagulant activity
Regression coefficient for PC:A
SE for regression coefficient
HGVS, Human Genome Variation Society; PC:A, PC anticoagulant activity; SE, standard error; SNP, single-nucleotide polymorphism; VIF, variance inflation factor. Multiple linear regression was used to evaluate the association between the three PROC gene SNPs and the PC:A levels. PC:A was determined in 69 carriers of the PROC c.574_576del variant and in 72 non-carriers. The PC:A value was loge-transformed for regression analysis. A high absolute value indicated that the corresponding polymorphism had a strong effect on PC:A, whereas a low absolute value indicated that the corresponding polymorphism had a weak effect on PC:A.
Table 3. Protein C (PC) anticoagulant activity in different genotypes
PC activity (U dL−1, mean ± SD)
SD, standard deviation. Each sample was tested in duplicate. dup, the PROC c.574_576dup wild-type allele; del, the PROC c.574_576del variant allele.
104.7 ± 11.8
101.9 ± 13.4
99.5 ± 8.9
100.8 ± 11.0
96.8 ± 9.0
94.2 ± 7.6
73.7 ± 6.8
72.1 ± 5.5
67.7 ± 6.7
Linkage analysis in the family of the only homozygote identified
Only one homozygote for the PROC c.574_576del variant was identified in this study. Family members were chosen for linkage analysis. The proband was a 48-year-old Chinese male who had experienced two episodes of confirmed VT. He was first hospitalized because of a spontaneous DVT in the left lower extremity at the age of 43 years. The second episode was a DVT in both lower extremities after knee joint trauma in the previous year. His older sister also suffered from DVT during the perinatal period 30 years before. However, five other relatives, who were heterozygotes for this variant, did not report thrombotic episodes. Hemostatic tests revealed that the proband had a low PC anticoagulant activity. Resequencing of the PROC gene did not show any other genetic abnormality except for the PROC c.574_576del variant in this family. This variant cosegregated completely with a low PC anticoagulant activity in the pedigree (Fig. S2). Moreover, the PC anticoagulant activity of the homozygote (proband) was only 42.5 ± 1.6 U dL−1, much lower than those of the heterozygotes, indicating an additive effect of the variant allele.
In vitro expression studies
As shown in Fig. 3A, the mean transcription level of mutant PC was approximately 1.31-fold higher than that of wild-type PC. However, the concentration of mutant PC was similar to that of wild-type PC (1.08-fold, P =0.413). The amidolytic activity of mutant PC in the culture media was 95.0% of that of wild-type PC, as measured with the chromogenic method. In contrast, the anticoagulant activity of mutant PC was reduced to 43.6% of that of wild-type PC. Mutant PC was found to be significantly less efficient than wild-type PC in prolonging the activated partial thromboplastin time (Fig. 3B). These results were consistent with those obtained with the PC anticoagulant activity tests in variant carriers and non-carriers described above.
PC is a vitamin K-dependent plasma anticoagulant that regulates blood coagulation by inactivating FVa and FVIIIa . Studies have shown that low PC activity caused by genetic mutations is linked to mild to severe VT incidents [37–39].
The PROC c.574_576del (p.Lys192del or p.Lys193del, rs199469469) variant in exon 7 will generate a transcript with an in-frame deletion of three nucleotides (AAG) and result in a PC translation product with a lysine deletion at position 192 or 193. The corresponding residue number in the mature protein is 150 or 151 (150Kdel or 151Kdel). Lys150 and Lys151 in the mature PC are highly conserved among mammals (Fig. S3). This variant was first described in three Japanese patients who suffered from PC deficiency in 1998 . In another study on PC and PS deficiencies in Japanese, the PROC c.574_576del variant was also identified in two of 85 DVT patients as well as in one of 30 healthy individuals . The researchers considered it to be a rare genetic mutation, and did not investigate whether it was a prevalent risk factor for VT in the general population. In this study, we found that the PROC c.574_576del variant was present in ∼ 2.36% (95% CI 1.60–3.13%) of the general Chinese population. The carrier rate was lower than that of FV Leiden (5–10%) and was similar to that of prothrombin G20210A (2–4%) . It is worth noting that, although the PC anticoagulant activities of variant carriers were lower than those of non-carriers, most of them were almost within the normal range (Fig. 2). Therefore, this PC variant should be considered as ‘a polymorphism’ rather than ‘a mutation’, and heterozygotes for this polymorphism should be considered as having ‘PC activity decreased’ rather than ‘PC deficiency’ .
In the coagulation screening stage, elevated FV activity was observed in PC-deficient individuals. This relationship might be explained by the fact that a deficiency in PC might result in inefficient and insufficient inactivation of its natural substrate, FV. This could lead to an imbalance between blood coagulation and anticoagulation, and a state of high FV activity in plasma.
In addition, PC activity determined with a clotting assay was different from that determined with a chromogenic method. A reasonable explanation for this is as follows. PC with a lysine deletion at position 150 or 151 adjacent to epidermal growth factor domains may interact with FVa, Ca2+, phospholipids or PS with relatively low efficiency, and result in a prolongation of the time needed to inactivate FVa completely. As a result, low activity caused by variants hampering the interaction of PC with its natural substrates and cofactors may not be identified with the chromogenic method [43–46]. Several such examples have been reported before [47,48].
Two SNPs (rs1799808 and rs1799809) in the promoter region of the PROC gene were reported to have a mild effect on plasma PC levels in white individuals. That is, the CC/GG genotype is associated with a slightly lower level of PC than the TT/AA genotype . Our linear regression model and genotype–phenotype analysis showed that the PROC c.574_576del variant had the strongest correlation with the decreased anticoagulant activity level of PC, suggesting that the low anticoagulant activity observed was mainly caused by this variant in the study population.
In conclusion, this study is the largest on common genetic risk factors for VT in the Chinese population. By the use of coagulation screening-related tests, resequencing, and a case–control study, we revealed, for the first time, that the common PROC c.574_576del variant was associated with both decreased PC anticoagulant activity and an increased risk of VT in the Chinese population. As this deletion variant was reported in several Japanese patients and one healthy individual 14 years ago, one might expect this polymorphism to be a common risk factor for VT in other Asian populations. Therefore, its prevalence in other Asians warrants further evaluation. The inclusion of this variant in routine thrombophilic detection may improve diagnosis and prevention strategies.
We are indebted to all of the participants in this study and the medical assistants of our hospital. Our special thanks go to the Department of Vascular Surgery (Union Hospital), the Ministry of Education Key Lab for Environment and Health (Tongji Medical College), and the co-workers at Shanghai Dongfang Hospital. We also thank X. Gao for kindly providing HEK-293 cells. We are grateful to C. Chen and L.-S. Ai for their expert technical assistance.
Disclosure of Conflict of Interests
This work was supported by the National Basic Scientific Research Program of China (973 Program, No. 2007 CB935803), the National Natural Sciences Foundation of China (No. 30825018), and the State Ministry of Health Key Clinical Construction Project (2010 No. 58).