Women may be at increased risk for venous thromboembolism (VTE) as compared with men. We studied the effects of genetic and biochemical markers of thrombophilia in women, in conjunction with other established risk factors for VTE.
The present retrospective case-control study was conducted in a thrombosis treatment programme at a large Toronto hospital. The cases were 129 women aged 16-79 years with objectively confirmed VTE. Age-matched control individuals were women who were free of venous thrombosis. Neither cases nor control individuals had known cardiovascular disease. Participants were interviewed regarding personal risk factors for VTE, including smoking, history of malignancy, pregnancy, and oestrogen or oral contraceptive use. Blood specimens were analyzed for common single nucleotide polymorphisms of prothrombin, factor V and methylenetetrahydrofolate reductase (MTHFR; C677T, A1298C and T1317C), and the A66G polymorphism for methionine synthase reductase (MTRR).Fasting plasma homocysteine was also analyzed.
Women with VTE were significantly more likely than female control individuals to carry the prothrombin polymorphism and the factor V polymorphism, or to have fasting hyperhomocysteinaemia. Homozygosity for the C677T MTHFR gene was not a significant risk factor for VTE, or were the A1298C or T1317C MTHFR homozygous variants. Also, the A66G MTRR homozygous state did not confer an increased risk for VTE.
Prothrombin and factor V polymorphisms increased the risk for VTE in women, independent from other established risk factors. Although hyperhomocysteinaemia also heightens this risk, common polymorphisms in two genes that are responsible for homocysteine remethylation do not. These findings are consistent with previous studies that included both men and women.
The development of both idiopathic and situational VTE remains multifactorial [
Published data implicate both the guanine-to-adenine nucleotide 20210 (G20210A) prothrombin and guanine-to-adenine nucleotide 1691 (G1691A) factor V gene polymorphisms in the development of VTE during pregnancy and the puerperium [
In order to better define the role of thrombophilic defects and other risk factors in the development of VTE in women, we conducted the present study. In addition to probable genetic risk factors [
We conducted a retrospective case-control study. Eligible cases included all women aged 16-79 years with objectively confirmed VTE seen at the University Health Network in Toronto from April 1996 to October 1999. Objective diagnostic testing comprised compression ultra-sonography or venography for deep vein thrombosis of the limb, ventilation/perfusion or contrast enhanced spiral computed tomography imaging for pulmonary embolism, and magnetic resonance or computed tomography imaging for intra-abdominal or intracranial venous thrombosis. We excluded those women whose VTE was related to a central venous catheter [
At the initial visit, a clinical nurse specialist interviewed cases using a standardized data collection form. An assessment for VTE risk factors included age at first VTE, history of recurrent VTE, history of malignancy within the past 5 years, current cigarette consumption, and immobilization or surgery within 3 months before the diagnosis of VTE. Presence of an 'oestrogen exposure state' was defined as either current use of an oral contraceptive agent, hormone replacement therapy or tamoxifen, or being pregnant or within 6 weeks postpartum. Information was also collected about the anatomical site of the VTE and the method of detection. Control group data were obtained using a standard questionnaire, administered by a trained research assistant during a face-to-face interview. The same demographic information was collected for cases and control individuals, with the exception of a history of recent immobilization or surgery, which was not assessed in the control group.
All laboratory analyses were performed with the investigator blinded to each woman's clinical diagnosis. Participants were instructed to fast for at least 8 h before plasma tHcy specimens were collected. Neither cases nor control individuals were known to be taking folate or vitamin B12 supplements at the time of blood specimen collection. Plasma tHcy was analyzed using high-performance liquid chromatography as previously described [
In the primary analysis, the association between the presence of VTE and each thrombophilic risk factor was evaluated using conditional logistic regression analysis. Crude and adjusted odds ratios (ORs) were estimated. The ORs were adjusted for specific covariates, which were defined
A predefined subgroup analysis was performed excluding cases and their respective control individuals with either immobilization or surgery within the past 3 months, and any case-control pairs in which either had had a malignancy within the past 5 years. The potential presence of another identifiable thrombophilia defect among the cases (eg protein S, protein C or antithrombin deficiency) was not accounted for in any of the analyses.
Genotype distributions and allele frequencies and equilibria were analyzed using Genetic Data Analysis version 1.0 (PO Lewis and D Zaykin, 2000, Sinauer Associates, Incorporated, Sunderland, MA, USA). Probabilities for allele frequencies were calculated using Fisher's exact test. For each disequilibrium (Hardy-Weinberg or pairwise linkage), the data program estimated the probability of obtaining the observed genotype distribution using 3600 permuted simulations generated by an allele-shuffling algorithm.
Baseline characteristics of cases and control individuals were compared using one-way analysis of variance for continuous variables, or the Χ2 test for categorical data. All
After initial review, 54 potential cases were excluded. Reasons for exclusion included the presence of cardiovascular disease (29 women), VTE related to central venous catheters (seven women), recurrent pregnancy loss, heparin-induced thrombocytopenia, superficial phlebitis or cellulitis (eight women), and lack of objective evidence to confirm the diagnosis of VTE (10 women). Thus, 129 cases, along with 129 age-matched control individuals, were included.
Table
Characteristics of 129 women with VTE and age-matched control individuals
Women who | Women who did not | Statistical comparison | |
experienced VTE | experience VTE | between cases | |
Characteristic | (129 cases) | (129 controls) | and controls |
Mean (SD) age (years) | 45.8 (17.5) | 40.9 (17.8) | |
Number (percentage) with deep vein thrombosis of the leg | 88 (68.2) | - | - |
Number (percentage) with deep vein thrombosis at another site | 17 (13.2) | - | - |
Number (%) with pulmonary embolism* | 32 (24.8) | - | - |
Number (%) with recurrent VTE | 33 (25.6) | - | - |
Number (%) with other thrombophilia defects† | 9 (7.0) | - | - |
Number (%) with immobilization or surgery within 3 months | 23 (17.8) | - | - |
Number (%) with malignancy within 5 years | 31 (24.0) | 8 (6.2) | |
Number (%) with oestrogen exposure state‡ | 44 (34.1) | 43 (33.3) | |
Number (%) current cigarette smokers | 17 (13.2) | 30 (23.2) | |
Mean (SD) fasting plasma total homocysteine (μmol/l) | 11.1 (5.6) | 6.5 (3.0) | |
Mean (SD) red-cell folate (nmol/l)$ | 1121.3 (566.1) | 981.9 (452.6) | |
Mean (SD) serum creatinine (μmol/l)$ | 82.2 (62.8) | 69.2 (10.3) |
*Describes individuals who either experienced pulmonary embolism in isolation or in combination with a deep vein thrombosis. †Includes either a positive test for antiphospholipid antibodies, the lupus anticoagulant, or a deficiency in protein C, protein S or antithrombin. ‡Defined as current use of an oral contraceptive agent, hormone replacement therapy or tamoxifen, or currently pregnant or within 6 weeks postpartum. $Complete data for serum creatinine and red-cell folate concentrations were only available for 80 case-control pairs. -, Data not applicable.
A nonstudy thrombophilia defect was identified in nine cases (7%). Four had positive anticardiolipin antibodies, and a fifth had a positive lupus anticoagulant. Of these five women, one was homozygous for both MTHFR C677T and A1298C, and heterozygous for the factor V polymorphism; one woman was homozygous for MTHFR A1298C; one woman was homozygous for MTRR A66G; and the remaining two women had no other identifiable defects. Three other women displayed mild functional protein S deficiency, of which one demonstrated homozy-gosity for both the MTHFR A1298C and MTRR A66G polymorphisms; one was homozygous for the MTHFR A1298C polymorphism; and the third was homozygous for the MTRR A66G polymorphism. One other woman was found to have antithrombin deficiency, but had no other detectable abnormalities.
Of the six loci examined (Table
Allele frequencies of the prothrombin, factor V, MTHFR and MTRR polymorphisms among 129 women with VTE and 129 matched control individuals
Gene polymorphism | ||||||||||||
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Prothrombin | Factor V | MTHFR | MTHFR | MTHFR | MTRR | |||||||
G20210A | G1691A | C677T | A1298C | T1317C | A66G | |||||||
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Allele | Cases | Controls | Cases | Controls | Cases | Controls | Cases | Controls | Cases | Controls | Cases | Controls |
-/- | 117 | 126 | 108 | 124 | 49 | 72 | 68 | 69 | 122 | 123 | 25 | 29 |
+/- | 11 | 3 | 19 | 5 | 61 | 44 | 49 | 49 | 6 | 5 | 62 | 65 |
+/+ | 1 | 0 | 2 | 0 | 19 | 13 | 12 | 11 | 1 | 1 | 42 | 35 |
Frequency | 0.050 | 0.012 | 0.089 | 0.019 | 0.384 | 0.271 | 0.283 | 0.275 | 0.031 | 0.027 | 0.566 | 0.523 |
Fisher's exact test |
0.02 | 0.0007 | 0.008 | 0.92 | 1.0 | 0.4 |
-/-, Wildtype; +/-, heterozygote; +/+, homozygote.
Among the participants, women with VTE were significantly more likely than control individuals to be carriers of the prothrombin polymorphism (9.3% versus 2.3%; adjusted OR 4.3, 95% confidence interval [CI] 1.1-17.4; Table
Prevalence and adjusted ORs for thrombophilia among women with VTE
Excluding women | |||||
with malignancy, | |||||
recent surgery or | |||||
All women | immobilization* | ||||
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Number (%) | Number (%) | ||||
of cases with | controls with | ||||
thrombophilia | thrombophilia | Crude OR | Adjusted OR† | Adjusted OR‡ | |
Thrombophilia factor | factor | factor | (95% CI) | (95% CI) | (95% CI) |
Prothrombin (++ or +/-) | 12 (9.3) | 3 (2.3) | 4.3 (1.2-15.6) | 4.3 (1.1-17.4) | 7.8 (1.0-64.2) |
Factor V (+/+ or +/-) | 21 (16.3) | 5 (3.9) | 4.8 (1.8-13.2) | 3.9 (1.4-10.8) | 3.6 (1.2-11.4) |
MTHFR C677T (+/+) | 19 (14.7) | 13 (10.1) | 1.5 (0.7-3.3) | 1.4 (0.6-3.1) | 1.1 (0.4-2.9) |
MTHFR 1298C (+/+) | 68 (52.7) | 69 (53.5) | 1.0 (0.6-1.6) | 1.0 (0.6-1.7) | 1.2 (0.6-2.3) |
MTRR A66G (+/+) | 42 (32.6) | 35 (27.1) | 1.3 (0.8-2.2) | 1.1 (0.6-2.2) | 0.9 (0.4-2.2) |
Any MTHFR (+/+) or A66G MTRR (+/+) | 90 (69.8) | 88 (68.2) | 1.1 (0.6-1.8) | 1.0 (0.6-2.0) | 1.1 (0.5-2.3) |
Fasting plasma hyperhomocysteinemia$ | 46 (35.6) | 5 (4.0) | 13.7 (5.2-36.0) | 17.8 (4.2-74.9) | 11.6 (2.7-50.5) |
*Excludes cases with immobilization or surgery within the past 3 months, or a malignancy within the past 5 years, and their respective control individuals. †Adjusted for cigarette smoking, oestrogen exposure state and recent history of cancer. ‡Adjusted for cigarette smoking and oestrogen exposure state. $Defined as a homocysteine concentration greater than the 95th centile value in the control group. +/+, Homozygous; +/-, heterozygous.
The MTRR A66G homozygous state was more common among cases (32.6%) than control individuals (27.1%), but not significantly (adjusted OR 1.1, 95% CI 0.6-2.2). Homozygosity for C677T MTHFR was no more frequent among cases (14.7%) than control individuals (10.1%; adjusted OR 1.4, 95% CI 0.6-3.1), and neither was the homozygous state for A1298C MTHFR (52.7% versus 53.5%; adjusted OR 1.0, 95% CI 0.6-1.7; Table
The mean fasting plasma tHcy was significantly higher among women with VTE (11.1 μmol/l) than control individuals (6.5 μmol/l; unpaired
Upon excluding cases with secondary causes for VTE (malignancy, immobilization or surgery), and their respective control individuals, 81 case-control pairs were available for analysis (Table
We found that the presence of the prothrombin and factor V gene polymorphisms and hyperhomocysteinaemia significantly increased the risk for VTE among women. When those with recent malignancy, surgery or immobilization were excluded, the risk was even greater for carriers of the prothrombin polymorphism. Although hyperhomocysteinaemia also increased the risk for VTE, this association was not found for the MTRR A66G or any of the three MTHFR gene polymorphisms.
Of the cases studied, 7% were found to have another thrombophilia defect, such as antiphospholipid antibodies or protein S deficiency, which was not controlled for in the present analysis. It is unlikely that the presence of such defects influenced the results, however, because they were uncommon, and eight out of nine of the defects were observed among women with the MTHFR or MTRR gene polymorphisms, or both, none of which independently conferred an increased risk for VTE.
The present study might have been limited by a referral bias among the cases, potentially exaggerating our risk estimates for VTE [
Published case-control studies of the risk for VTE in the presence of polymorphisms of the prothrombin, factor V and MTHFR C677T genes, as well as fasting hyperhomocysteinaemia
Prevalence of | ||||||
thrombophilia defect (%) | ||||||
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Thrombophilia defect | Reference | Population | VTE type | Cases | Controls | Risk estimate (95% CI) |
Prothrombin gene (+/+ or +/-) | [ |
Men and women | Any VTE | 4.6 | 1.0 | ORc 4.8 (1.5-19.8) |
[ |
Men and women | Any VTE | 14.2 | 4.6 | ORc 3.4 (2.2-5.5) | |
[ |
Men and women | Any VTE | 10.2 | 2.8 | ORa 4.0 (1.9-8.5) | |
[ |
Men and women | Any VTE | 18.5 | 5.4* | ORa 3.6 (1.8-7.3) | |
[ |
Men and women | Any DVT | 7.1 | 1.8 | ORa 3.8 (1.1-13.2) | |
[ |
Men and women | First DVT | 6.2 | 2.3 | ORc 2.8 (1.4-5.6) | |
[ |
Men and women | First DVT | 15.9 | 2.3 | ORa 8.7 (3.8-21.4) | |
[ |
Pregnant women | Any VTE | 16.9 | 1.3 | RRa 15.2 (4.2-52.6) | |
[ |
Pregnant women | Any VTE | 31.0 | 4.2 | ORa 10.2 (4.0-25.9) | |
Present study | Women | Any VTE | 9.3 | 2.3 | ORa 4.3 (1.1-17.4) | |
Factor V gene (+/+ or +/-) | [ |
Men and women | Any VTE | 12.5 | 1.4 | ORc 2.7 (1.5-4.9) |
[ |
Men and women | Any VTE | 18.2 | 5.1 | ORc 4.2 (2.7-6.4) | |
[ |
Men and women | Any VTE | 53.0 | 3.3 | ORc 17.4 (11.4-93.0) | |
[ |
Men and women | Any VTE | 26.0 | 7.6 | ORc 4.3 (2.3-8.1) | |
[ |
Men and women | Any VTE | 19.5 | 3.5 | ORa 6.5 (3.5-12.5) | |
[ |
Men and women | Any VTE | 40.1 | 3.9† | ORa 16.3 (8.5-31.3) | |
[ |
Men and women | DVT and PE | 14.8 | 5.3 | ORc 4.2 (1.5-10.3) | |
[ |
Men and women | Any DVT | 19.5 | 2.9 | ORc 8.0 (4.5-14.2) | |
[ |
Men and women | Any DVT | 28.0 | 11.0 | ORa 3.1 (1.7-5.5) | |
[ |
Men and women | Any DVT | 23.5 | 6.1 | ORc 4.7 (1.5-15.0) | |
[ |
Men and women | First DVT | 21.1 | 3.2 | ORa 7.8 (3.9-17.1) | |
[ |
Pregnant women | Any VTE | 43.7 | 7.7 | RRa 9.3 (5.1-16.9) | |
[ |
Pregnant women | Any VTE | 23.8 | 1.9 | ORc 16.3 (4.8-54.9) | |
Present study | Women | Any VTE | 16.3 | 3.9 | ORa 3.9 (1.4-10.8) | |
MTHFR gene (+/+) | [ |
Men and women | Any VTE | 10.0 | 13.0 | ORc 0.7 (0.5-1.0) |
[ |
Men and women | Any VTE | 28.2 | 17.7 | ORc 1.8 (1.2-2.9) | |
[ |
Men and women | Any VTE | 11.5 | 15.0 | ORc 0.7 (0.4-1.3) | |
[ |
Men and women | Any VTE | 12.7 | 12.3 | ORa 1.0 (0.6-1.7) | |
[ |
Men and women | Any VTE | 22.8 | 14.3 | ORa 2.1 (1.2-3.7) | |
[ |
Men and women | Any VTE | 12.3‡ | 13.0 | ORc 0.9 (0.4-2.4) | |
[ |
Men and women | Any VTE | 8.0 | 6.4 | ORc 1.4 (0.5-4.0) | |
[ |
Men and women | Any VTE | 12.3 | 13.1 | ORc 0.9 (0.4-2.3) | |
[ |
Men and women | Any DVT | 7.4 | 10.6 | ORc 0.7 (0.2-2.2) | |
[ |
Men and women | Any DVT | 25.6 | 18.1 | ORc 1.7 (1.1-2.5) | |
[ |
Men and women | Any DVT | 10.0 | 9.9 | ORc 1.0 (0.7-1.5) | |
[ |
Men and women | First DVT | 20.5 | 21.0 | ORa 1.0 (0.7-1.2) | |
[ |
Pregnant women | Any VTE | 28.6 | 16.0 | ORc 2.1 (1.0-4.5) | |
Present study | Women | Any VTE | 14.7 | 10.1 | ORa 1.4 (0.6-3.1) | |
Fasting hyperhomocysteinaemia | [ |
Men and women | Any VTE | 12.5 | 4.7 | ORp 3.0 (2.1-4.2)$ |
[ |
Men and women | Any VTE | 16.0 | 5.0 | ORc 3.6 (1.8-7.3) | |
Present study | Women | Any VTE | 35.6 | 4.0 | ORa 17.8 (4.2-74.9) |
*Describes the +/- genotype. †Describes the +/+ genotype. ‡Describes the prevalence of the MTHFR C677T +/+ state among individuals with factor V. $Comprises pooled data from nine observational studies. DVT, deep vein thrombosis; +/+, homozygous; +/-, heterozygous; ORa, adjusted OR; ORc, crude OR; ORp, pooled OR; RRa, adjusted relative risk.
Although the rate of fasting hyperhomocysteinaemia found among our control individuals (4.0%) was consistent with those of other studies (range 4.7-5.0%; Table
A meta-analysis of 23 observational studies [
If hyperhomocysteinaemia is a true causative risk factor for a first [
Others have evaluated the influence of nongenetic factors on the risk for VTE. For example, in a post-trial analysis by the Heart Estrogen Replacement Study (HERS) investigators [
Clinicians face a dilemma in deciding which women should be screened for these and other thrombophilia markers. It would seem both imprudent and costly to investigate those who develop VTE in the presence of active malignancy, because anticoagulant therapy is likely to be continued indefinitely [
CI = confidence interval; MTHFR = methylenetetrahydrofolate reductase; MTRR = methionine synthase reductase; OR = odds ratio; RH = relative hazard; tHcy = total homocysteine; VTE = venous thromboembolism.
Dr Cole is supported in part by grant #T4340 from the Ontario Heart and Stroke Foundation. Special thanks to Dr Murray Urowitz for his assistance in recruiting the controls for this study.