Factor V gene G1691A mutation, prothrombin gene G20210A mutation, and MTHFR gene C677T mutation are not risk factors for pulmonary thromboembolism in Chinese population

Thrombosis Research 106 (2002) 7 – 12

Regular Article

Factor V gene G1691A mutation, prothrombin gene G20210A mutation, and MTHFR gene C677T mutation are not risk factors for pulmonary thromboembolism in Chinese population Yanhui Lu a, Yanfen Zhao a, Guozhang Liu b, Xiaoling Wang c, Zhihong Liu a, Baiping Chen a, Rutai Hui d,* a Division of Cardio-Pulmonary Circulation, Cardiovascular Institute and Fu Wai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100037, People’s Republic of China b Division of Hypertension, Cardiovascular Institute and Fu Wai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100037, People’s Republic of China c Division of Population Genetics and Prevention, Cardiovascular Institute and Fu Wai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100037, People’s Republic of China d Sino – German Laboratory for Molecular Medicine, Cardiovascular Institute and Fu Wai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 167 Beilishi Street, XiCheng District, Beijing 100037, People’s Republic of China

Received 5 October 2001; received in revised form 8 February 2002; accepted 10 April 2002 Accepting Editor: N. Tandon

Abstract A mutation in coagulant factor V gene, a substitution in the 3V untranslated region of prothrombin gene, and a variant in 5,10methylenetetrahydrofolate reductase (MTHFR) gene have been reported to be related to venous thromboembolism in Caucasians, but this relationship remains in debate in other populations. In this case – control study, we aimed to determine the prevalence of these three mutations in the Chinese population and investigate whether they are associated with pulmonary thromboembolism. The subjects consisted of 90 patients with pulmonary thromboembolism and 143 age- and sex-matched healthy controls. Traditional risk factors for venous thrombosis and pulmonary thromboembolism were investigated as well. We found that the frequencies of varicose vein, phlebitis, trauma, and surgery were significantly higher in cases than in controls. Of all subjects, none was a carrier of coagulant factor V gene G1691A mutation or prothrombin gene G 20210A mutation, which are common point mutations in Caucasians, while the frequency of MTHFR gene 677T allele exceeded 55% in cases (56.7%), controls (55.2%), and all subjects (55.8%) studied. The homozygous mutation rate of MTHFR gene was over 30% in both cases (33.3%) and controls (32.2%), but no significant difference was found in genotype distribution (v2 = 0.100, P = 0.951). Model fitting using logistic regression did not find that MTHFR gene C677T mutation was associated with pulmonary thromboembolism, consistent with the results of stratification analysis. In conclusion, coagulant factor V gene G1691A mutation and prothrombin G20210A mutation are very rare, while MTHFR C677T mutation is very common, and all of them are not associated with pulmonary thromboembolism in the Chinese population. D 2002 Elsevier Science Ltd. All rights reserved. Keywords: Pulmonary thromboembolism; Coagulant factor V gene Leiden mutation; MTHFR gene C677T mutation; Prothrombin gene G20210A mutation

1. Introduction

Abbreviations: APC, activated protein C; APCR, resistance to activated protein C; FVa, activated coagulant factor V; FV Leiden, factor V gene Leiden; MTHFR, 5,10-methylenetetrahydrofolate reductase. * Corresponding author. Tel.: +86-10-6833-3902; fax: +86-10-68331730. E-mail address: [email protected] (R. Hui).

Venous thromboembolism, including venous thrombosis and pulmonary thromboembolism, constitutes a major health problem. As the important complication of venous thrombosis, pulmonary thromboembolism is one of the most common causes of death occurring in the acute care hospitals, accounting for 15% of all deaths in these situations. Conditions associated with increased risk of venous throm-

0049-3848/02/$ - see front matter D 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 9 - 3 8 4 8 ( 0 2 ) 0 0 0 6 4 - 6

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Y. Lu et al. / Thrombosis Research 106 (2002) 7–12

Table 1 Sequences of primers, length of PCR product, and restriction digestion (bp) Mutation

Sequence of primer

PCR product

Restriction enzyme

Wild type

Heterozygosity

Mutated homozygosity

FV Leiden

5VTGCCCAGTGCTTAACAAGACCA3V 5VTGTTATCACACTGGTGCTAA3V 5VTCTAGAAACAGTTGCCTGGC3V 5VATAGCACTGGGAGCATTGAAGC3V 5VTGAAGGAGAAGGTGTCTGCGGGA3V 5VAGGACGGTGCGGTGAGAGTG3V

267

Mnl I

163/67/37

200/163 67/37

200/67

345

Hind III

345

345/322/23

322/23

198

Hinf I

198

198/175/23

175/23

PT20210 G/A MTHFR 677C/T

FV—factor V; PT—prothrombin; MTHFR—5,10-methylenetetrahydrofolate reductase.

boembolism include not only environmental factors, but also genetic predisposition. Inherited resistance activated protein C (APCR) was found in 1993 [1], and it was established that APCR could be explained in 90– 95% of cases by the G1691A mutation in coagulant factor V gene (FV Leiden mutation) [2]. This mutation results in the Arg506Gln substitution, which is the key site in the cleavage of activated coagulant factor V (FVa) by activated protein C (APC). The mutated FVa maintains the normal coagulation activity, but resists anticoagulant function of APC. Individuals with this mutation exhibit the hypercoagulable state and an increased risk of venous thromboembolism. Many studies have confirmed that FV Leiden mutation increases the risk of venous thromboembolism, but there seems to be a discrepancy in the prevalence of this mutation among different races. Another inherited risk factor for venous thromboembolism, a G/A transition at nucleotide 20210 in the 3V untranslated region of prothrombin gene, was identified in 1996 [3]. The mutation was reported to be associated with a higher prothrombin clotting activity with a 2.7-fold increased risk of venous thromboembolism. Several reports confirmed similar findings in Caucasians [4 –6], but results from other races were different [7,8]. Hyperhomocysteinemia has been established as a risk factor for both arterial and venous thrombosis. Moderate elevation of plasma homocysteine concentration could be caused either by deficiencies in vitamin B12, vitamin B6, and folic acid, or by genetic deficiencies of related metabolic enzymes. A common point mutation in 5,10-methylenetetrahydrofolate reductase (MTHFR) gene identified by Frosst et al. [9], a C/T substitution at nucleotide 677, changes a highly conserved alanine into a valine residue and results in an elevated plasma level of homocysteine. But there still

exists a controversy as to whether MTHFR gene C677T mutation is associated with an increased risk of venous thromboembolism. Almost all of the epidemiological evidence evaluating the association between these three mutations and venous thromboembolism pertains to the white population. However, there are obvious racial differences in the prevalence of these mutations and the correlation of these mutations with pulmonary thromboembolism remains inconclusive. The purpose of this present case – control study was to determine the prevalence of FV Leiden mutation, prothrombin gene G20210A mutation, and MTHFR gene C677T mutation in the Chinese population and to investigate whether these genetic variants are associated with pulmonary thromboembolism.

2. Materials and methods 2.1. Subjects Ninety patients with pulmonary thromboembolism and 143 sex- and age-matched healthy controls were recruited from September 1998 to December of 2000 in Fu Wai Hospital, Chinese Academy of Medical Sciences. The mean ages for cases and controls were 49 and 48 years, respectively, ranging from 23 to 81 years for cases and from 21 to 79 years for controls. Forty-six patients with pulmonary thromboembolism (51.1%; 46/90) also had evidence of deep venous thrombosis. Pulmonary thromboembolism was diagnosed by lung ventilation/perfusion scan, or ultrafast CT and medical history. Traditional risk factors for venous thrombosis and pulmonary thromboembolism were recorded, including history of trauma, surgery, oral contraceptive,

Table 2 Distributions of risk factors for VTE in cases and controls (%) Group

Number

Family history of VTE

Family history of CVD

Cigarette smoking

Case Control

90 143

2.2 0

35.5 38.5

42.2 46.2

VTE—venous thromboembolism; CVD—cardiovascular disease. * P < 0.005. ** P < 0.001.

Alcohol drinking 27.7 24.5

Oral contraceptive

Trauma

Surgery

Phlebitis varicose vein

2.2 2.8

15.6 * 4.2

24.4* * 2.1

18.9* * 1.4

Y. Lu et al. / Thrombosis Research 106 (2002) 7–12

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Table 3 Distribution of genotypes and alleles of MTHFR gene C677T mutation

Case Control Total

Number

Genotype (n/F) CC

CT

TT

C

T

90 143 233

18 (0.200) 31 (0.217) 49 (0.210)

42 (0.467) 66 (0.461) 108 (0.464)

30 (0.333) 46 (0.322) 76 (0.326)

78 (0.433) 128 (0.448) 206 (0.442)

102 (0.567) 158 (0.552) 260 (0.558)

v2 P

Allele (n/F)

0.100 0.951

0.090 0.764

varicose vein, phlebitis, cigarette smoking, alcohol drinking, and family history of venous thromboembolism and cardiovascular diseases. Cases were excluded if embolism was due to fat, air, tumor, and amniotic fluid. 2.2. DNA preparation and genotyping for mutations Venous blood (5 ml) anticoagulated with EDTA (2% ethylene diaminetetraacetic acid – sodium salt) was extracted with phenol –chloroform to isolate genomic DNA. Polymerase chain reaction (PCR) was employed to amplify the target fragments. All amplifications (25 Al) were completed in a reaction mixture containing 2.0 mmol/l MgCl2, 50 mmol/l KCl, 10 mmol/l Tris – HCl (pH 8.3), 200 Al/l of each dNTP, 1 U of Taq DNA polymerase, and 20 pmol/l of each primer (Sangon, Shanghai, China). Table 1 shows the information on sequences of primers, PCR products, and digestion sites by restriction enzymes. The G1691A mutation in exon 10 of coagulant factor V gene was detected by the loss of a cleavage site for MnlI (New England Biolaboratories, Beverly, MA, USA). A 267-bp fragment was amplified from genomic DNA [2]. The fragments measuring 163, 67, and 37 bp for allele 1691G, and 200 and 67 bp for allele 1691A, were separated on 12% polyacrylamide gel electrophoresis. A 345bp fragment including the 3V untranslated region of prothrombin gene, where the G20210A transition is located, was amplified as described previously [3]. PCR products subjected to a restriction digestion with HindIII (Promega, Madison, WI, USA) were analyzed on 3% agarose gel and visualized with ethidium bromide. The C677T mutation in MTHFR gene was tested as described by Frosst et al. [9]. PCR products were subjected to restriction digestion by Hinf I (Promega), and the digested fragments were separated on 3% agarose gel and visualized with ethidium bromide.

2.3. Statistical analysis Ratios of traditional risk factors for venous thromboembolism were calculated and compared using Chi-square test between cases and controls. Allele frequencies were deduced from the genotype frequencies. Hardy – Weinberg equilibrium was tested in cases, controls, and the combined samples, respectively. Allele and genotype frequencies were compared between cases and controls by Chi-square test. Genotype associations with qualitative phenotypes (case/ control status) were tested by logistic regression analysis. Odds ratios were computed from regression parameters. We used separate logistic regression model to assess the relationship between pulmonary thromboembolism and MTHFR gene C677T mutation [10]. The genotypes are denoted as TT, TC, and CC. We analyzed MTHFR gene C677T mutation by considering the expression of allele 677T either as dominant (with scores of 0 for CC and 1 for both TT and TC) or as recessive model (with scores of 1 for TT and 0 for both TC and CC). Additionally, we fitted a logistic model with scores of 0 for CC genotype, 1 for TC genotype, and 2 for TT genotype. This model assumes that the presence of an additional allele 677T is associated with a multiplicative increase in pulmonary thromboembolism risk [10]. The significance level for statistical tests was taken to be 0.05.

3. Results 3.1. The basic characteristic of cases and controls In cases and controls, the distribution of traditional risk factors for venous thrombosis and pulmonary thromboembolism is shown in Table 2. The frequencies of varicose vein, phlebitis, trauma, and surgery were significantly higher in

Table 4 Thrombotic risk for MTHFR gene C677T mutation under different models TT + CT vs. CC Case Control OR (95% CI) P

72 112 0.903 (0.471 – 1.734) 0.760

TT vs. CT + CC 18 31

30 46 0.949 (0.541 – 1.663) 0.853

TT vs. CT vs. CC 60 97

30 46 0.976 (0.535 – 1.780) 0.936

42 66

18 31

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Y. Lu et al. / Thrombosis Research 106 (2002) 7–12

cases than those in controls, and there were no significant differences between cases and controls in history of oral contraceptives, personal history of cigarette smoking and alcohol drinking, and family history of venous thromboembolism and cardiovascular diseases.

surgery (within 1 month)], patients with isolated pulmonary thromboembolism (n = 44), and patients with both pulmonary thromboembolism and venous thrombosis (n = 46). We did not find any significant difference in the frequencies of genotypes between subgroups and controls (Table 5).

3.2. The prevalence of these three mutations 4. Discussion As shown in Table 3, of all subjects recruited, none was a carrier of FV Leiden mutation or prothrombin gene 20210G/A substitution. The frequencies of allele 677T in MTHFR gene in cases and controls were 56.7% and 55.2% (v2 = 0.09, P = 0.763), respectively, and the genotype distribution was in Hardy – Weinberg equilibrium. In cases and controls, the frequencies of homozygous mutation in MTHFR gene were 33.3% and 32.2%, respectively, but no significant difference was found in genotype distribution between two groups. 3.3. Relationship between MTHFR gene C677T mutation and pulmonary thromboembolism under different models To investigate whether MTHFR gene C677T mutation is associated with pulmonary thromboembolism, logistic regression analysis was performed under recessive (with scores of 1 for TT genotype and 0 for both CC and CT), dominant (with scores of 0 for CC and 1 for TC and TT), and multiplicative models (with scores of 0 for CC, 1 for TC, and 2 for TT). We did not find an association between MTHFR gene C677T mutation and pulmonary thromboembolism. The allele 677T did not increase the risk of pulmonary thromboembolism under any genetic model (Table 4). 3.4. Stratification analysis In order to assess whether MTHFR gene C677T mutation has a different effect on patients with or without environmental risk factors and whether MTHFR gene C677T mutation exerts a different influence on patients with isolated pulmonary thromboembolism and patients with both pulmonary thromboembolism and venous thrombosis, we divided the cases into four subgroups, such as patients with (n = 28) or without (n = 62) environmental risk factors [medical history of phlebitis, varicose vein, and recent trauma or Table 5 Thrombotic risk for MTHFR gene C677T mutation in subgroups Groups

Number (n)

Genotype (n) CC

CT

TT

C

T

Control subgroup of case With other risk factor Without other risk factor With venous thrombosis Without venous thrombosis

143 28 62 46 44

31 4 14 7 11

66 14 28 21 21

46 10 20 18 12

128 22 56 35 43

158 34 68 57 45

90

18

42

30

78

102

Total cases

Allele (n)

Our results indicate that varicose vein, phlebitis, trauma, and surgery are risk factors for pulmonary thromboembolism in the Chinese population. Oral contraceptives did not contribute to the risk of pulmonary thromboembolism in our study. This phenomenon is probably related to the seldom use of oral contraceptives in the Chinese population in the past decades. Family history of venous thromboembolism has been confirmed to be the major risk factor for first and recurrent events of venous thromboembolism in Caucasians. In our study, however, only two patients had family history of venous thromboembolism, which suggests that familial pulmonary thromboembolism should not be common in the Chinese population. Our results indicate that the risk profile of venous thromboembolism in the Chinese population is different from Caucasians. The initial report of APCR was of a patient with recurrent deep venous thrombosis whose plasma, unlike the normal response, did not produce a prolongation in the activated partial thromboplastin test (APTT)-based clotting assay when purified APC was added. Linkage study and subsequent investigation revealed a G/A substitution at nucleotide 1691 in coagulant factor V gene (FV Leiden mutation). Further study confirmed that FV Leiden mutation is in 90– 95% of cases of APCR. Overall, heterozygosity for FV Leiden mutation is associated with a 5- to 10-fold increased risk of venous thromboembolism, and homozygosity with a 50- to 100-fold increased risk [2,11]. But there is a significant geographic variation in the prevalence of FV Leiden mutation. FV Leiden mutation appears very frequently in the Indo-European population with reported frequency ranging from 2% to 15% and very rare in indigenous populations from Asian, African, Australian, Greenland, and American origin. Even within the same country, significant variation in prevalence can occur. This highlights the importance of establishing the frequency of FV Leiden mutation in common, ethnically derived population rather than using political boundaries. In our study, we did not find FV Leiden mutation in all of the 233 subjects. Our result is very similar with those of other Asian studies from Thailand [7], Japan [12], Korea [13], and Taiwan [8,14,15], and further supports that FV Leiden mutation is very rare in Asians and not a genetic risk factor for pulmonary thromboembolism. The low prevalence of FV Leiden mutation outside of European-derived populations suggests that a founding effect from a single ancient mutation should have occurred. Recent migration from Europe probably accounts for the distribution of FV Leiden mutation in the western

Y. Lu et al. / Thrombosis Research 106 (2002) 7–12

world rather than recurrent mutations occurring at the wellestablished mutation hot spot. High prothrombin clotting activity has been considered to be associated with an increased risk of venous thromboembolism. Carriers of prothrombin gene G20210A mutation tend to have higher prothrombin levels than noncarriers. The prevalence of prothrombin gene G20210A mutation in healthy controls is 1.8% among the Swedish [16], 0.7 – 2.6% among the English [17,18], 1.2– 2.3% among Netherlanders [3,19], 1.8% among Australians [20], 3.5% among the Spanish [21], and 2.3% among Italians [22]. It ranges from 3% to 18% in Caucasian patients with venous thromboembolism. Many studies indicate that prothrombin gene G20210A mutation is an independent risk factor for venous thromboembolism in Caucasian. But studies from other races or regions are completely different. The prevalence of this mutation is very low or close to 0% among Asians, Africans, and native Americans, indicating that the mutation could not be considered as a risk factor for venous thromboembolism in these populations. Of all subjects in our study, we did not find prothrombin gene G20210A mutation. The results are similar with those from Asians, Africans, and native Americans, indicating that the mutation does not influence the incidence of pulmonary thromboembolism in the Chinese population. The MTHFR gene C677T mutation has been shown to correlate with a reduced MTHFR activity and an increased thermolability in lymphocyte pellets. Individuals with homozygous (TT) mutation have higher plasma levels of homocysteine than heterozygosity (TC) or those without the mutation (CC). Several epidemiological studies have found a direct association between elevated plasma homocysteine levels and venous thromboembolism. Up to now, the results on the direct relationship between MTHFR gene C677T mutation and venous thromboembolism are controversial. Frosst et al. reported the rate of homozygosity for MTHFR gene C677T mutation in French –Canadian subjects to be 12%. Other investigators have reported that this rate was about 5% in whites, 12 – 15% in populations of European, Middle Eastern, and Japanese origin, 5.4% in Finnish populations, 5.2% in Dutch populations, and 1.4% in African – Americans. However, no clear association of the homozygous mutation with thrombosis could be demonstrated in patients with venous thromboembolism. In most studies on patients with venous thromboembolism, the frequency of homozygosity for the mutation in cases was not higher than that in controls, indicating that the homozygous mutation is not associated with venous thromboembolism. In our present study, the frequency of homozygosity for the mutation in healthy controls is significantly higher (33.2%) than that from Caucasians and Africans, which is contrary to that of FV Leiden mutation and prothrombin G20210A mutation. But there is no significant difference in allele and genotype distribution in MTHFR gene C677T mutation between cases and controls. Both model fitting and stratification analysis

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did not find that MTHFR gene C677T mutation is associated with pulmonary thromboembolism in the Chinese population, suggesting that MTHFR gene C677T mutation should be a common polymorphism, but not a genetic risk factor for venous thromboembolism in the Chinese population. This result is similar to that obtained in the Taiwan population [23]. In summary, our study supports the growing body of evidence which indicates that FV Leiden mutation and prothrombin G20210A mutation are considerably rare in the Chinese population. However, the frequency of 677T allele for MTHFR gene is significantly higher in the Chinese population than that in Caucasians, which suggests that MTHFR gene C677T mutation should be a common polymorphism in the Chinese population and does not increase the risk of pulmonary thromboembolism. Additionally, this study indicates that the risk profile of pulmonary thromboembolism in the Chinese population is decidedly different from that in Caucasians.

Acknowledgements This work was supported by the Division of CardioPulmonary Circulation and Sino – German Laboratory. We thank all of the persons who helped us collect data or finish the experimental work.

References [1] Dahlback B, Carlsson M, Svensson PJ. Familial thrombophilia due to a previously unrecognised mechanism characterized by poor anticoagulant response to activated protein C: prediction of a cofactor to activated protein C. Proc Natl Acad Sci USA 1993;90:1004 – 8. [2] Bertina RM, Koeleman BPC, Koster T, Rosendaal FR, Dirven RJ, deRonde H, van der Velden PA, Reitsma PH. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994;369:64 – 7. [3] Poort SR, Rosendaal FR, Reitsma PH, Bertina RM. A common genetic variation in the 3V-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and increase in venous thrombosis. Blood 1996;88:3698 – 703. [4] Leroyer C, Mercier B, Oger E, Chenu E, Abgrall JF, Ferec C, Mottier D. Prevalence of 20210A allele of the prothrombin gene in venous thromboembolism patients. Thromb Haemostasis 1998;80:49 – 51. [5] Hillarp A, Zoller B, Svensson P, Dahldack B. The 20210A allele of the prothrombin gene is a common risk factor among Swedish outpatients with verified deep venous thrombosis. Thromb Haemostasis 1997;78:990 – 2. [6] Margaglione M, Brancaccio V, De Lucia D, Martinelli I, Ciampa A, Grandone E, Di Minno G. Inherited thrombophilic risk factors and venous thromboembolism: distinct role in peripheral deep venous thrombosis and pulmonary embolism. Chest 2000;118(5):1234 – 6. [7] Angchaisuksiri P, Pingsuthiwong S, Aryuchai K, Busabaratana M, Sura T, Atichartakarn V, Sritara P. Prevalence of the G1691A mutation in the factor V gene (factor V Leiden) and the G20210A prothrombin gene mutation in the Thai population. Am J Hematol 2000;65:119 – 22. [8] Shen MC, Lin JS, Tsay W. Protein C and protein S deficiencies are the most important risk factors associated with thrombosis in Chinese

12

[9]

[10]

[11]

[12]

[13]

[14]

[15] [16]

Y. Lu et al. / Thrombosis Research 106 (2002) 7–12 venous thrombophilic patients in Taiwan. Thromb Res 2000;99(5): 447 – 52. Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, Boers GJH, den Heijer M, Kluijtmans LAJ, van den Heuvel LP, Rozen R. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet 1995; 10:111 – 3. Dilly A, Austin H, Hooper WC, Lally C, Ribeiro MJA, Wenger NK, Silva V, Rawlins P, Evatt B. Relation of three genetic traits to venous thrombosis in an African – American population. Am J Epidemiol 1998;147:30 – 5. Rosendaal FR, Koster T, Vandenbroucke JP, Reitsma PH. High risk of thrombosis in patients homozygous for factor V Leiden (activated protein C resistance). Blood 1995;85:1504 – 8. Fujimura H, Kambayashi J, Monden M, Kato H, Miyata T. Coagulation factor V Leiden mutation may have a racial background. Thromb Haemostasis 1995;74:1381 – 3. Kim TW, Kim WK, Lee JH, Kim SB, Kim SW, Suh C, Lee KH, Lee JS, Seo EJ, Chi HS, Kim SH. Low prevalence of activated protein C resistance and coagulation factor V Arg506 to Gln mutation among Korean patients with deep vein thrombosis. J Korean Med Sci 1998; 13:587 – 90. Shen MC, Lin JS, Tsay W. 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 1997; 87:377 – 85. Ho CH, Chau WK, Hsu HC. Causes of venous thrombosis in fifty Chinese patients. Am J Hematol 2000;63(2):74 – 8. Hillarp A, Zoller B, Svensson PJ, Dahlback B. The 20210 A allele of the prothrombin gene is a common risk factor among Swedish outpatients with verified deep venous thrombosis. Thromb Haemostasis 1997;78:990 – 2.

[17] Cumming AM, Keeney S, Salden A, Bhavnani M, Shwe KH, Hay CRM. The prothrombin gene G20210A variant: prevalence in a UK anticoagulant clinic population. Br J Haematol 1997;98:353 – 5. [18] Makris M, Preston FE, Beauchamp NJ, Cooper PC, Daly ME, Hampton KK, Bayliss P, Peake IR, Miller GJ. Co-inheritance of the 20210A allele of the prothrombin gene increase the risk of thrombosis in subjects with familial thrombophilia. Thromb Haemostasis 1997; 78:1426 – 9. [19] Doggen CMJ, Cats VM, Bertina RM, Rosendaal FR. Interaction of coagulation defects and cardiovascular risk factors. Increased risk of myocardial infarction associated with factor V Leiden or prothrombin 20210A. Circulation 1998;97:1037 – 41. [20] Renner W, Koppel H, Hoffmann C, Schallmoser K, Stanger O, Toplak H, Wascher TC, Pilger E. Prothrombin G20210A, factor V Leiden, and factor XIII Val34Leu: common mutations of blood coagulation factors and deep vein thrombosis in Austria. Thromb Res 2000; 99:35 – 9. [21] Vargas M, Soto I, Pinto CR, Urgelles MF, Batalla A, Rodriguez-Reguero J, Cortina A, Alvarez V, Coto E. The prothrombin 20210A allele and the factor V Leiden are associated with venous thrombosis but not with early coronary artery disease. Blood Coagulation Fibrinolysis 1999;10:39 – 41. [22] Cattaneo M, Chantarangkul V, Taioli E, Santos JH, Tagliabue L. The G20210A mutation of the prothrombin gene in patients with previous first episodes of deep-vein thrombosis: prevalence and association with factor V G1691A, methylenetetrahydrofolate reductase C677T and plasma prothrombin levels. Thromb Res 1999;93:1 – 8. [23] Lin JS, Shen MC, Tsai W, Lin B. The prevalence of C677T mutation in the methylenetetrahydrofolate reductase gene and its association with venous thrombophilia in Taiwanese Chinese. Thromb Res 2000; 97:89 – 94.