Hyperhomocysteinemia and Risk of First Venous Thrombosis: The Influence of (Unmeasured) Confounding Factors

Hyperhomocysteinemia and Risk of First Venous Thrombosis: The Influence of (Unmeasured)... Abstract Meta-analyses have reported a 2- to 3-fold increased risk of venous thrombosis (VT) in individuals with hyperhomocysteinemia. However, confounding factors were generally not considered. In contrast, randomized trials of homocysteine-lowering therapy and VT risk have been negative. We investigated whether hyperhomocysteinemia was associated with VT in the Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis (MEGA) case-control study (1999–2004) from the Netherlands (1,689 cases and 1,726 controls), taking into account measured and unmeasured confounders. We compared patients with population controls to estimate odds ratios using unconditional logistic regression and adjusted for various potential confounders. We matched patients to their partners to additionally adjust for unmeasured confounders (e.g., lifestyle factors) using conditional logistic regression. We found that elevated homocysteine concentrations were not associated with an increased risk for VT when comparing patients with population controls, either as a continuous variable (odds ratio = 1.00, 95% confidence interval: 0.99, 1.01), in terms of 0.7-mg/L increase (odds ratio = 0.99, 95% confidence interval: 0.93, 1.05), or within different homocysteine categories. We obtained similar results when patients were compared with their partners. Stratification by sex, deep vein thrombosis, pulmonary embolism, provoked VT, and unprovoked VT also provided no evidence of an association. In conclusion, after extensive adjustments for confounding, hyperhomocysteinemia was not associated with an increased risk of venous thrombosis in this study. homocysteine, homocystinuria, venous thrombosis, vitamin supplementation Venous thrombosis (VT) accounts for an important burden of disease in the world (1). A systematic review of the literature reported an overall annual incidence of VT ranging from 0.75 to 2.69 per 1,000 individuals in Western Europe, North America, Australia, and Latin America (1). Multiple risk factors for VT have been reported; one is hyperhomocysteinemia, but its causal association with VT has remained controversial (2). Homocysteine is an intermediate amino acid in the metabolism of methionine and cysteine. It was first linked to cardiovascular disease and VT in 1969 (3). Three meta-analyses demonstrated a modest association of increased VT risk per 0.7-mg/L (5 μmol/L) higher homocysteine concentration or hyperhomocysteinemia (4–6). However, the available evidence makes it difficult to discern whether the association between hyperhomocysteinemia and VT is a causal or spurious phenomenon. First, proper adjustment for age, sex, and many lifestyle-related factors that could confound the relationship was not feasible in the meta-analyses because most studies did not report an adjusted odds ratio due to small sample sizes. For instance, in the 1990s, case-control studies usually included no more than 75 cases with VT with similarly few controls (4). The numbers of cases and controls since then have also been fairly small, with a maximum number of 397 cases with VT and 585 controls in a study from 2001 (7). Second, previous studies defined hyperhomocysteinemia differently (i.e., homocysteine concentrations above the 95th percentile or mean plus 2 standard deviations calculated from the distribution in control groups), and the cutoff levels for presence of hyperhomocysteinemia, when mentioned, ranged from >1.1 mg/L to >3.4 mg/L (4, 8). Third, these meta-analyses did not exclude publication bias, which could have resulted in an overestimation of the risk. And fourth, some studies have suggested that the association of homocysteine with VT is visible only in subgroups in which conventional risk factors for VT are absent (9). Hyperhomocysteinemia can be treated with B-vitamin supplementation (folic acid, pyridoxine, and cobalamin), but several studies (including 2 clinical trials) failed to demonstrate any benefit from such homocysteine-lowering therapy in the prevention of VT (10–12). It has been argued that the biological mechanism through which homocysteine increases the risk of vascular disease is more complex than an increase in homocysteine only (13). Additionally, these clinical trials present some limitations that preclude the conclusion that the reported association between homocysteine and VT was related to the effect of confounders. For example, the follow-up time (average of less than 4 years) might have been too short to show a risk reduction in VT incidence among individuals treated with homocysteine-lowering treatment. In addition, the generalizability of results might not be applicable to the general population, because the 2 clinical trials that evaluated the benefit of homocysteine-lowering therapy were conducted either among patients with a prior history of VT or among those with a higher risk of cardiovascular disease (10, 11). We decided to report on the association among various concentrations of homocysteine and VT in a single, large, population-based case-control study to examine whether the association between hyperhomocysteinemia and first VT event persisted after adjustment for both measured and unmeasured confounding factors. The analysis included 1,689 patients with a first VT event, either deep vein thrombosis (DVT) of the leg or pulmonary embolism, and 1,726 control subjects from the Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis (MEGA) study. This study enabled us to compare associations with VT for various homocysteine cutoff levels in various subgroups in which we could adjust for both measured and unmeasured confounding factors by comparing patients with population-derived (using random-digit dialing (RDD)) controls and with the patients’ partners, which formed the 2 control groups of the study. METHODS Study population The design of the MEGA case-control study is described elsewhere (14). In short, MEGA is a large, population-based case-control study that recruited 4,956 cases with a first diagnosis of deep VT or pulmonary embolism, from 6 anticoagulation clinics in the Netherlands, between March 1999 and September 2004. The diagnosis of DVT was confirmed by Doppler ultrasonography. The diagnosis of pulmonary embolism was made by ventilation-perfusion lung scan, spiral computed tomography, or angiogram. Two different control groups were invited to participate, patients’ partners (n = 3,295) and controls identified using RDD (n = 3,000). The latter control group was recruited from January 2002 through September 2004. These participants were also between 18 and 70 years of age, with no previous history of VT. For logistical reasons, blood sampling was performed for participants included up to June 2002. Data collection and definitions The date of diagnosis was assigned as the index date for patients and their partner controls, and for RDD controls, the index date was the date of signing informed consent. All participants were asked to complete a questionnaire about demographic characteristics, lifestyle, risk factors for VT, and cardiovascular diseases. Body mass index was calculated using self-reported weight and height. Smoking habits were classified as current smoker, previous smoker, or nonsmoker. Risk factors for provoked VT (prior surgery, plaster cast immobilization, trauma, hospitalization in the previous 3 months, long-distance travel in the preceding 2 months, or malignancy at the time of or in the 5 years before the index date) were obtained with the questionnaire. Women were also asked about the use of hormone therapy (hormonal replacement therapy or oral-contraceptive use). Participants were asked about cardiovascular disease (self-reported previous myocardial infarction or ischemic stroke) and statin use. Individuals involved in sports activities at least once a week were considered to be practicing regular sports activity. Laboratory measurements Fasting blood samples were taken from patients approximately 3 months after ending anticoagulation therapy. In patients who continued their anticoagulation therapy, blood was sampled 1 year after the index date. Partner controls provided their fasting blood sample along with their partners (patients). RDD controls were invited for a fasting blood draw after returning their questionnaires. Blood samples were collected in tubes with citrate anticoagulant to prevent increases in homocysteine concentration (15). Total homocysteine plasma concentrations were measured in a central laboratory (Laboratory of Pediatrics and Neurology in Nijmegen, the Netherlands) by an automated high-performance liquid chromatography method with reverse-phase and fluorescent detection (Gilson 232-401 sample processor (Gilson Inc., Middleton, Wisconsin), Spectra Physics 8800 solvent delivery system, and Spectra Physics LC 304 fluorometer (Spectra-Physics, San Jose, California)). Inclusion and exclusion criteria Of the 4,956 patients, we excluded 182 women who were pregnant at the index date or within the previous 3 months. These women were excluded because guidelines recommend folic acid during pregnancy and pregnancy itself affects risk (16). Next, we excluded 1,467 patients whose vitamin consumption information was missing or who were using vitamin-B therapy, because trials have shown that vitamin B intake lowers plasma homocysteine concentrations without affecting VT risk (10, 11). Finally, we excluded 1,618 patients who were included in the study after June 2002 because blood samples were no longer drawn from this date onward. This left us with 1,689 patients. Of these patients, 787 had a partner who fulfilled the inclusion criteria and was willing to participate, so 787 matched pairs remained. After application of the same exclusion criteria on the RDD control group, 939 RDD control participants were included in the analysis. Statistical analysis The analysis of plasma homocysteine concentrations was carried out in 2 separate ways, as a continuous (per 0.13-mg/L increase or 1-μmol/L increase) variable and using categories. Plasma concentrations of homocysteine were divided into categories of 0.26 mg/L. Values lower than 1.6 mg/L were allocated to the lowest (reference) category, and values higher than 2.3 mg/L to the highest category. Next, we performed an analysis for extremely high versus normal concentrations of homocysteine using the following categories: in mg/L, 2.30–3.19, 3.20–6.49, and ≥6.50. We set 2.3 mg/L as the cutoff value because a previous analysis in the Leiden Thrombophilia Study (LETS) showed that VT risk was increased only for concentrations over 2.3 mg/L (18 μmol/L) (17). The analysis was repeated with 0.7-mg/L intervals because a previous meta-analysis observed a 1.3- to 1.6-fold increased risk per 0.7-mg/L (5 μmol/L) increase of homocysteine (4). Odds ratios with 95% confidence intervals were calculated as an estimate of the relative risk of VT for the different concentrations of homocysteine. When the analysis was made for all patients and RDD controls, we used unconditional logistic regression, adjusting for age and sex. Analyses were further adjusted for body mass index, smoking, statin use, history of arterial cardiovascular disease, and regular sports activities. Because some studies indicate a different relationship between elevated homocysteine concentration and VT in women and men (12, 18, 19), we additionally performed an analysis stratified by sex. Within this sex-stratified analysis, we additionally adjusted for hormone therapy in women. Because partners of patients are likely to resemble the patients in health behavior more than do random-digit dialing controls, we performed a 1:1-matched analysis by conditional logistic regression, which adjusts for associations within matched pairs. This method provides adjustment for all unmeasured factors for which couples tend to be similar (20). The analysis is conditional given that many clinical characteristics of controls, who are individually matched to the patients, are likely to be similar to patients’ characteristics. In this analysis, all aforementioned potential confounding factors were also adjusted for. Although using partners as controls results in most controls having the opposite sex as their matched case, one can adjust for sex in a partner-matched case-control study by allowing for sex with an indicator variable (19). Nevertheless, we also performed an analysis where only men were compared with men and women with women. Furthermore, odds ratios were calculated to estimate risk for VT associated with homocysteine concentrations of ≥2.3 mg/L, using as reference range homocysteine <2.3 mg/L for patients with provoked or unprovoked VT, and patients with deep vein thrombosis, pulmonary embolism, or both in subgroup analyses. All statistical analyses were performed using SPSS for Windows, release 20.0 (SPSS Inc., Chicago, Illinois). Conditional logistic regression was performed using the COXREG procedure (21). Because many studies on homocysteine report homocysteine concentrations in μmol/L instead of mg/L, we decided to show our analyses on homocysteine concentrations in both mg/L (in the main article) and in μmol/L (in Web Tables 1–6, available at https://academic.oup.com/aje) RESULTS A total of 3,415 participants (1,689 patients, 787 partner controls, and 939 RDD controls) were included in the study (Figure 1). The main characteristics of the participants are presented in Tables 1 and 2. The mean age was 49 years in patients, 50 years in partner controls, and 48 years in RDD controls (all in age range 18–70 years). Homocysteine concentrations above 2.3 mg/L were present in 261 (15%) patients, 93 (12%) partner controls, and 117 (13%) RDD controls. In general, in the category of homocysteine concentrations lower than 1.6 mg/L, more people of young age, female sex, or engaged in sports activities were present than in the other categories. As expected, VT risk factors were more often present in patients than in controls. Figure 1. View largeDownload slide Selection of patients with first venous thrombosis (VT) and controls in the Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis (MEGA) study, the Netherlands, 1999–2004. A) Selection of patients with history of VT; B) selection of partner controls; C) selection of controls identified using random-digit dialing (RDD). Figure 1. View largeDownload slide Selection of patients with first venous thrombosis (VT) and controls in the Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis (MEGA) study, the Netherlands, 1999–2004. A) Selection of patients with history of VT; B) selection of partner controls; C) selection of controls identified using random-digit dialing (RDD). Table 1. Clinical Characteristics of Patients With Venous Thrombosis Included in the Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis Case-Control Study, the Netherlands, 1999–2004 Characteristica  Homocysteine Concentration, mg/L  Overall  <1.60  1.60–1.79  1.80–2.09  2.10–2.29  ≥2.30  No.  %  No.  %  No.  %  No.  %  No.  %  No.  %  Total  1,689  888  250  187  103  261  Men  850  50  370  42  143  57  123  66  58  56  156  60  Age at enrollment, yearsb  49 (18–70)  47 (18–70)  50 (19–70)  50 (18–70)  52 (21–70)  52 (19–70)  BMIb,c  27 (17–58)  27 (17–58)  27 (18–47)  27 (18–46)  27 (18–40)  27 (18–49)  Malignancy  89  5  39  4  8  3  13  7  10  10  19  7  Hormone use (in women)  514  62  343  67  56  52  42  67  24  56  49  48  Risk factors for venous thrombosis                           Provokedd  863  51  497  56  107  43  91  49  51  50  117  45   Unprovoked  818  49  387  44  143  57  95  51  51  50  142  55  Arterial cardiovascular risk factors                           Previous smoking  520  31  263  30  76  31  62  33  29  28  90  35   Current smoking  584  35  278  31  92  37  68  37  41  40  105  40   Self-reported prior CVDe  55  4  15  2  13  6  10  6  3  3  14  6   Statin use  62  4  33  4  11  4  4  2  1  1  13  5   Regular sports activity  512  34  292  37  82  37  55  33  23  25  60  27  Characteristica  Homocysteine Concentration, mg/L  Overall  <1.60  1.60–1.79  1.80–2.09  2.10–2.29  ≥2.30  No.  %  No.  %  No.  %  No.  %  No.  %  No.  %  Total  1,689  888  250  187  103  261  Men  850  50  370  42  143  57  123  66  58  56  156  60  Age at enrollment, yearsb  49 (18–70)  47 (18–70)  50 (19–70)  50 (18–70)  52 (21–70)  52 (19–70)  BMIb,c  27 (17–58)  27 (17–58)  27 (18–47)  27 (18–46)  27 (18–40)  27 (18–49)  Malignancy  89  5  39  4  8  3  13  7  10  10  19  7  Hormone use (in women)  514  62  343  67  56  52  42  67  24  56  49  48  Risk factors for venous thrombosis                           Provokedd  863  51  497  56  107  43  91  49  51  50  117  45   Unprovoked  818  49  387  44  143  57  95  51  51  50  142  55  Arterial cardiovascular risk factors                           Previous smoking  520  31  263  30  76  31  62  33  29  28  90  35   Current smoking  584  35  278  31  92  37  68  37  41  40  105  40   Self-reported prior CVDe  55  4  15  2  13  6  10  6  3  3  14  6   Statin use  62  4  33  4  11  4  4  2  1  1  13  5   Regular sports activity  512  34  292  37  82  37  55  33  23  25  60  27  Abbreviations: BMI, body mass index; CVD, cardiovascular disease. a Data were missing for some participants in some subgroups. b Values are expressed as mean (range). c Weight (kg)/height (m2). d Provoked: previous surgery, malignancy, immobilization, trauma, plaster cast, oral contraceptive/hormonal replacement therapy, or recent travel. e CVD denotes self-reported myocardial infarction or ischemic stroke. View Large Table 1. Clinical Characteristics of Patients With Venous Thrombosis Included in the Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis Case-Control Study, the Netherlands, 1999–2004 Characteristica  Homocysteine Concentration, mg/L  Overall  <1.60  1.60–1.79  1.80–2.09  2.10–2.29  ≥2.30  No.  %  No.  %  No.  %  No.  %  No.  %  No.  %  Total  1,689  888  250  187  103  261  Men  850  50  370  42  143  57  123  66  58  56  156  60  Age at enrollment, yearsb  49 (18–70)  47 (18–70)  50 (19–70)  50 (18–70)  52 (21–70)  52 (19–70)  BMIb,c  27 (17–58)  27 (17–58)  27 (18–47)  27 (18–46)  27 (18–40)  27 (18–49)  Malignancy  89  5  39  4  8  3  13  7  10  10  19  7  Hormone use (in women)  514  62  343  67  56  52  42  67  24  56  49  48  Risk factors for venous thrombosis                           Provokedd  863  51  497  56  107  43  91  49  51  50  117  45   Unprovoked  818  49  387  44  143  57  95  51  51  50  142  55  Arterial cardiovascular risk factors                           Previous smoking  520  31  263  30  76  31  62  33  29  28  90  35   Current smoking  584  35  278  31  92  37  68  37  41  40  105  40   Self-reported prior CVDe  55  4  15  2  13  6  10  6  3  3  14  6   Statin use  62  4  33  4  11  4  4  2  1  1  13  5   Regular sports activity  512  34  292  37  82  37  55  33  23  25  60  27  Characteristica  Homocysteine Concentration, mg/L  Overall  <1.60  1.60–1.79  1.80–2.09  2.10–2.29  ≥2.30  No.  %  No.  %  No.  %  No.  %  No.  %  No.  %  Total  1,689  888  250  187  103  261  Men  850  50  370  42  143  57  123  66  58  56  156  60  Age at enrollment, yearsb  49 (18–70)  47 (18–70)  50 (19–70)  50 (18–70)  52 (21–70)  52 (19–70)  BMIb,c  27 (17–58)  27 (17–58)  27 (18–47)  27 (18–46)  27 (18–40)  27 (18–49)  Malignancy  89  5  39  4  8  3  13  7  10  10  19  7  Hormone use (in women)  514  62  343  67  56  52  42  67  24  56  49  48  Risk factors for venous thrombosis                           Provokedd  863  51  497  56  107  43  91  49  51  50  117  45   Unprovoked  818  49  387  44  143  57  95  51  51  50  142  55  Arterial cardiovascular risk factors                           Previous smoking  520  31  263  30  76  31  62  33  29  28  90  35   Current smoking  584  35  278  31  92  37  68  37  41  40  105  40   Self-reported prior CVDe  55  4  15  2  13  6  10  6  3  3  14  6   Statin use  62  4  33  4  11  4  4  2  1  1  13  5   Regular sports activity  512  34  292  37  82  37  55  33  23  25  60  27  Abbreviations: BMI, body mass index; CVD, cardiovascular disease. a Data were missing for some participants in some subgroups. b Values are expressed as mean (range). c Weight (kg)/height (m2). d Provoked: previous surgery, malignancy, immobilization, trauma, plaster cast, oral contraceptive/hormonal replacement therapy, or recent travel. e CVD denotes self-reported myocardial infarction or ischemic stroke. View Large Table 2. Clinical Characteristics of Controls Included in the Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis Case-Control Study, the Netherlands, 1999–2004 Characteristica  Homocysteine Concentration, mg/L  Overall  <1.60  1.60–1.79  1.80–2.09  2.10–2.29  ≥2.30  No.  %  No.  %  No.  %  No.  %  No.  %  No.  %  Partners of Patients  Total  787  449  117  76  52  93  Men  386  49  196  44  59  50  44  58  32  62  55  59  Age at enrollment, yearsb  50 (18–70)  49 (18–70)  51 (28–68)  50 (26–70)  54 (30–70)  50 (21–70)  BMIb,c  27 (17–52)  26 (17–44)  26 (19–48)  27 (20–42)  26 (19–40)  25 (18–45)  Malignancy  10  1  4  1  2  2  2  3  1  2  1  1  Hormone use (in women)  78  20  59  24  9  17  4  13  3  15  3  8  Risk factors for venous thrombosis                           Provokedd  112  14  78  18  14  13  8  11  6  12  6  7   Unprovoked  661  86  366  82  98  87  66  89  46  88  85  93  Arterial cardiovascular risk factors                           Previous smoking  241  31  140  31  39  34  22  29  13  26  27  29   Current smoking  230  29  125  28  28  24  25  33  17  33  35  38   Self-reported prior CVDe  18  3  10  2  2  2  1  2  1  2  4  5   Statin use  43  6  26  6  7  6  3  4  3  6  4  4   Regular sports activity  263  38  165  41  33  32  23  35  12  27  30  37  Controls Identified by Random-Digit Dialing  Total  939  518  154  96  54  117  Men  491  52  241  47  89  58  57  59  28  52  76  65  Age at enrollment, yearsb  48 (18–70)  47 (18–70)  49 (20–70)  50 (20–70)  52 (23–70)  50 (19–69)  BMIb,c  25 (16–43)  25 (16–40)  26 (19–43)  26 (17–39)  26 (20–39)  25 (18–37)  Malignancy  20  2  11  2  3  2  2  2  2  4  2  2  Hormone use (% in women)  145  33  98  36  18  29  9  23  5  19  15  37  Risk factors for venous thrombosis                           Provokedd  195  21  126  25  26  17  12  13  9  17  22  19   Unprovoked  733  79  386  75  125  83  83  87  45  83  94  81  Arterial cardiovascular risk factors                           Previous smoking  289  31  150  29  49  32  37  39  17  32  36  31   Current smoking  279  30  147  29  39  26  29  30  18  33  46  40   Self-reported prior CVDe  26  3  14  3  3  2  4  4  1  2  4  3   Statin use  79  8  40  8  13  8  10  10  3  6  13  11   Regular sports activity  396  48  233  52  68  49  36  44  22  44  37  34  Characteristica  Homocysteine Concentration, mg/L  Overall  <1.60  1.60–1.79  1.80–2.09  2.10–2.29  ≥2.30  No.  %  No.  %  No.  %  No.  %  No.  %  No.  %  Partners of Patients  Total  787  449  117  76  52  93  Men  386  49  196  44  59  50  44  58  32  62  55  59  Age at enrollment, yearsb  50 (18–70)  49 (18–70)  51 (28–68)  50 (26–70)  54 (30–70)  50 (21–70)  BMIb,c  27 (17–52)  26 (17–44)  26 (19–48)  27 (20–42)  26 (19–40)  25 (18–45)  Malignancy  10  1  4  1  2  2  2  3  1  2  1  1  Hormone use (in women)  78  20  59  24  9  17  4  13  3  15  3  8  Risk factors for venous thrombosis                           Provokedd  112  14  78  18  14  13  8  11  6  12  6  7   Unprovoked  661  86  366  82  98  87  66  89  46  88  85  93  Arterial cardiovascular risk factors                           Previous smoking  241  31  140  31  39  34  22  29  13  26  27  29   Current smoking  230  29  125  28  28  24  25  33  17  33  35  38   Self-reported prior CVDe  18  3  10  2  2  2  1  2  1  2  4  5   Statin use  43  6  26  6  7  6  3  4  3  6  4  4   Regular sports activity  263  38  165  41  33  32  23  35  12  27  30  37  Controls Identified by Random-Digit Dialing  Total  939  518  154  96  54  117  Men  491  52  241  47  89  58  57  59  28  52  76  65  Age at enrollment, yearsb  48 (18–70)  47 (18–70)  49 (20–70)  50 (20–70)  52 (23–70)  50 (19–69)  BMIb,c  25 (16–43)  25 (16–40)  26 (19–43)  26 (17–39)  26 (20–39)  25 (18–37)  Malignancy  20  2  11  2  3  2  2  2  2  4  2  2  Hormone use (% in women)  145  33  98  36  18  29  9  23  5  19  15  37  Risk factors for venous thrombosis                           Provokedd  195  21  126  25  26  17  12  13  9  17  22  19   Unprovoked  733  79  386  75  125  83  83  87  45  83  94  81  Arterial cardiovascular risk factors                           Previous smoking  289  31  150  29  49  32  37  39  17  32  36  31   Current smoking  279  30  147  29  39  26  29  30  18  33  46  40   Self-reported prior CVDe  26  3  14  3  3  2  4  4  1  2  4  3   Statin use  79  8  40  8  13  8  10  10  3  6  13  11   Regular sports activity  396  48  233  52  68  49  36  44  22  44  37  34  Abbreviations: BMI, body mass index; CVD, cardiovascular disease. a Data were missing for some participants in some subgroups. b Values are expressed as mean (range). c Weight (kg)/height (m2). d Provoked: previous surgery, malignancy, immobilization, trauma, plaster cast, oral contraceptive/hormonal replacement therapy, or recent travel. e CVD denotes self-reported myocardial infarction or ischemic stroke. View Large Table 2. Clinical Characteristics of Controls Included in the Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis Case-Control Study, the Netherlands, 1999–2004 Characteristica  Homocysteine Concentration, mg/L  Overall  <1.60  1.60–1.79  1.80–2.09  2.10–2.29  ≥2.30  No.  %  No.  %  No.  %  No.  %  No.  %  No.  %  Partners of Patients  Total  787  449  117  76  52  93  Men  386  49  196  44  59  50  44  58  32  62  55  59  Age at enrollment, yearsb  50 (18–70)  49 (18–70)  51 (28–68)  50 (26–70)  54 (30–70)  50 (21–70)  BMIb,c  27 (17–52)  26 (17–44)  26 (19–48)  27 (20–42)  26 (19–40)  25 (18–45)  Malignancy  10  1  4  1  2  2  2  3  1  2  1  1  Hormone use (in women)  78  20  59  24  9  17  4  13  3  15  3  8  Risk factors for venous thrombosis                           Provokedd  112  14  78  18  14  13  8  11  6  12  6  7   Unprovoked  661  86  366  82  98  87  66  89  46  88  85  93  Arterial cardiovascular risk factors                           Previous smoking  241  31  140  31  39  34  22  29  13  26  27  29   Current smoking  230  29  125  28  28  24  25  33  17  33  35  38   Self-reported prior CVDe  18  3  10  2  2  2  1  2  1  2  4  5   Statin use  43  6  26  6  7  6  3  4  3  6  4  4   Regular sports activity  263  38  165  41  33  32  23  35  12  27  30  37  Controls Identified by Random-Digit Dialing  Total  939  518  154  96  54  117  Men  491  52  241  47  89  58  57  59  28  52  76  65  Age at enrollment, yearsb  48 (18–70)  47 (18–70)  49 (20–70)  50 (20–70)  52 (23–70)  50 (19–69)  BMIb,c  25 (16–43)  25 (16–40)  26 (19–43)  26 (17–39)  26 (20–39)  25 (18–37)  Malignancy  20  2  11  2  3  2  2  2  2  4  2  2  Hormone use (% in women)  145  33  98  36  18  29  9  23  5  19  15  37  Risk factors for venous thrombosis                           Provokedd  195  21  126  25  26  17  12  13  9  17  22  19   Unprovoked  733  79  386  75  125  83  83  87  45  83  94  81  Arterial cardiovascular risk factors                           Previous smoking  289  31  150  29  49  32  37  39  17  32  36  31   Current smoking  279  30  147  29  39  26  29  30  18  33  46  40   Self-reported prior CVDe  26  3  14  3  3  2  4  4  1  2  4  3   Statin use  79  8  40  8  13  8  10  10  3  6  13  11   Regular sports activity  396  48  233  52  68  49  36  44  22  44  37  34  Characteristica  Homocysteine Concentration, mg/L  Overall  <1.60  1.60–1.79  1.80–2.09  2.10–2.29  ≥2.30  No.  %  No.  %  No.  %  No.  %  No.  %  No.  %  Partners of Patients  Total  787  449  117  76  52  93  Men  386  49  196  44  59  50  44  58  32  62  55  59  Age at enrollment, yearsb  50 (18–70)  49 (18–70)  51 (28–68)  50 (26–70)  54 (30–70)  50 (21–70)  BMIb,c  27 (17–52)  26 (17–44)  26 (19–48)  27 (20–42)  26 (19–40)  25 (18–45)  Malignancy  10  1  4  1  2  2  2  3  1  2  1  1  Hormone use (in women)  78  20  59  24  9  17  4  13  3  15  3  8  Risk factors for venous thrombosis                           Provokedd  112  14  78  18  14  13  8  11  6  12  6  7   Unprovoked  661  86  366  82  98  87  66  89  46  88  85  93  Arterial cardiovascular risk factors                           Previous smoking  241  31  140  31  39  34  22  29  13  26  27  29   Current smoking  230  29  125  28  28  24  25  33  17  33  35  38   Self-reported prior CVDe  18  3  10  2  2  2  1  2  1  2  4  5   Statin use  43  6  26  6  7  6  3  4  3  6  4  4   Regular sports activity  263  38  165  41  33  32  23  35  12  27  30  37  Controls Identified by Random-Digit Dialing  Total  939  518  154  96  54  117  Men  491  52  241  47  89  58  57  59  28  52  76  65  Age at enrollment, yearsb  48 (18–70)  47 (18–70)  49 (20–70)  50 (20–70)  52 (23–70)  50 (19–69)  BMIb,c  25 (16–43)  25 (16–40)  26 (19–43)  26 (17–39)  26 (20–39)  25 (18–37)  Malignancy  20  2  11  2  3  2  2  2  2  4  2  2  Hormone use (% in women)  145  33  98  36  18  29  9  23  5  19  15  37  Risk factors for venous thrombosis                           Provokedd  195  21  126  25  26  17  12  13  9  17  22  19   Unprovoked  733  79  386  75  125  83  83  87  45  83  94  81  Arterial cardiovascular risk factors                           Previous smoking  289  31  150  29  49  32  37  39  17  32  36  31   Current smoking  279  30  147  29  39  26  29  30  18  33  46  40   Self-reported prior CVDe  26  3  14  3  3  2  4  4  1  2  4  3   Statin use  79  8  40  8  13  8  10  10  3  6  13  11   Regular sports activity  396  48  233  52  68  49  36  44  22  44  37  34  Abbreviations: BMI, body mass index; CVD, cardiovascular disease. a Data were missing for some participants in some subgroups. b Values are expressed as mean (range). c Weight (kg)/height (m2). d Provoked: previous surgery, malignancy, immobilization, trauma, plaster cast, oral contraceptive/hormonal replacement therapy, or recent travel. e CVD denotes self-reported myocardial infarction or ischemic stroke. View Large Odds ratios for VT with different fasting homocysteine concentrations are presented in Table 3, for comparisons between patients and RDD controls, and Table 4 for comparisons between patients and partner controls. Overall, the age and sex-adjusted odds ratio for fasting homocysteine concentration in the comparison of patients with RDD controls was 1.01 (95% confidence interval (CI): 1.00, 1.02) when using plasma homocysteine concentrations as a continuous (per 0.13-mg/L increase) variable and 1.04 (95% CI: 0.98, 1.10) in terms of 0.7-mg/L increase, and 1.30 (95% CI: 1.02, 1.67) when the concentrations of ≥2.3 mg/L were compared with concentrations <1.6 mg/L. In the analysis of concentrations of ≥2.3 mg/L relative to concentrations of <1.6 mg/L, the odds ratio was 1.30 (95% CI; 1.02, 1.67) adjusted for age and sex, 1.27 (95% CI: 0.98,1.64) additionally adjusting for body mass index and smoking, 1.28 (95% CI: 0.99,1.65) after adding statin use, 1.19 (95% CI: 0.91,1.55) after adding cardiovascular disease, and 1.02 (95% CI: 0.77,1.34) after adding sports activities. Results from the 1:1-matched analysis between patients and partner controls resembled those from patients versus RDD controls (Table 4). In the analysis stratified by sex, on patients and RDD controls, crude and fully adjusted odds ratios remained close to unity (Table 5). Table 3. Risk of Venous Thrombosis According to Categories of Homocysteine Concentrations in All Patients and Random-Digit Dialing Controls, Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis, the Netherlands, 1999–2004 Homocysteine Concentration, mg/L  All Patients  RDD Controls  Model 1a  Model 2b  Model 3c  Model 4d  Model 5e  No.  %  No.  %  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  <1.60  888  53  518  55  1.00  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.60–1.79  250  15  154  16  0.95  0.76, 1.20  0.92  0.72, 1.16  0.91  0.72, 1.16  0.9  0.70, 1.14  0.86  0.66, 1.11  1.80–2.09  187  11  96  10  1.15  0.88, 1.51  1.11  0.84, 1.47  1.86  0.82, 1.44  1.03  0.77, 1.37  1.08  0.79, 1.48  2.10–2.29  103  6  54  6  1.10  0.78, 1.57  1.01  0.71, 1.45  0.97  0.68, 1.39  0.97  0.67, 1.39  0.92  0.62, 1.34  ≥2.30  261  15  117  13  1.30  1.02, 1.67  1.27  0.98, 1.64  1.28  0.99, 1.65  1.19  0.91, 1.55  1.02  0.77, 1.34  Continuous          1.01  1.00, 1.02  1.00  0.99, 1.02  1.01  0.99, 1.02  1.00  0.99, 1.01  1.00  0.99, 1.01  Per 0.7-mg/L increase          1.04  0.98, 1.10  1.03  0.97, 1.09  1.03  0.97, 1.09  1.02  0.96, 1.07  0.99  0.93, 1.05  Homocysteine Concentration, mg/L  All Patients  RDD Controls  Model 1a  Model 2b  Model 3c  Model 4d  Model 5e  No.  %  No.  %  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  <1.60  888  53  518  55  1.00  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.60–1.79  250  15  154  16  0.95  0.76, 1.20  0.92  0.72, 1.16  0.91  0.72, 1.16  0.9  0.70, 1.14  0.86  0.66, 1.11  1.80–2.09  187  11  96  10  1.15  0.88, 1.51  1.11  0.84, 1.47  1.86  0.82, 1.44  1.03  0.77, 1.37  1.08  0.79, 1.48  2.10–2.29  103  6  54  6  1.10  0.78, 1.57  1.01  0.71, 1.45  0.97  0.68, 1.39  0.97  0.67, 1.39  0.92  0.62, 1.34  ≥2.30  261  15  117  13  1.30  1.02, 1.67  1.27  0.98, 1.64  1.28  0.99, 1.65  1.19  0.91, 1.55  1.02  0.77, 1.34  Continuous          1.01  1.00, 1.02  1.00  0.99, 1.02  1.01  0.99, 1.02  1.00  0.99, 1.01  1.00  0.99, 1.01  Per 0.7-mg/L increase          1.04  0.98, 1.10  1.03  0.97, 1.09  1.03  0.97, 1.09  1.02  0.96, 1.07  0.99  0.93, 1.05  Abbreviations: BMI, body mass index; CI, confidence interval; OR, odds ratio. a Model 1 adjusted for age and sex. b Model 2 adjusted for age, sex, BMI, and smoking. c Model 3 adjusted for age, sex, BMI, smoking, and statin use. d Model 4 adjusted for age, sex, BMI, smoking, statin use, and cardiovascular disease. e Model 5 adjusted for age, sex, BMI, smoking, statin use, cardiovascular disease, and sports activity. Table 3. Risk of Venous Thrombosis According to Categories of Homocysteine Concentrations in All Patients and Random-Digit Dialing Controls, Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis, the Netherlands, 1999–2004 Homocysteine Concentration, mg/L  All Patients  RDD Controls  Model 1a  Model 2b  Model 3c  Model 4d  Model 5e  No.  %  No.  %  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  <1.60  888  53  518  55  1.00  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.60–1.79  250  15  154  16  0.95  0.76, 1.20  0.92  0.72, 1.16  0.91  0.72, 1.16  0.9  0.70, 1.14  0.86  0.66, 1.11  1.80–2.09  187  11  96  10  1.15  0.88, 1.51  1.11  0.84, 1.47  1.86  0.82, 1.44  1.03  0.77, 1.37  1.08  0.79, 1.48  2.10–2.29  103  6  54  6  1.10  0.78, 1.57  1.01  0.71, 1.45  0.97  0.68, 1.39  0.97  0.67, 1.39  0.92  0.62, 1.34  ≥2.30  261  15  117  13  1.30  1.02, 1.67  1.27  0.98, 1.64  1.28  0.99, 1.65  1.19  0.91, 1.55  1.02  0.77, 1.34  Continuous          1.01  1.00, 1.02  1.00  0.99, 1.02  1.01  0.99, 1.02  1.00  0.99, 1.01  1.00  0.99, 1.01  Per 0.7-mg/L increase          1.04  0.98, 1.10  1.03  0.97, 1.09  1.03  0.97, 1.09  1.02  0.96, 1.07  0.99  0.93, 1.05  Homocysteine Concentration, mg/L  All Patients  RDD Controls  Model 1a  Model 2b  Model 3c  Model 4d  Model 5e  No.  %  No.  %  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  <1.60  888  53  518  55  1.00  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.60–1.79  250  15  154  16  0.95  0.76, 1.20  0.92  0.72, 1.16  0.91  0.72, 1.16  0.9  0.70, 1.14  0.86  0.66, 1.11  1.80–2.09  187  11  96  10  1.15  0.88, 1.51  1.11  0.84, 1.47  1.86  0.82, 1.44  1.03  0.77, 1.37  1.08  0.79, 1.48  2.10–2.29  103  6  54  6  1.10  0.78, 1.57  1.01  0.71, 1.45  0.97  0.68, 1.39  0.97  0.67, 1.39  0.92  0.62, 1.34  ≥2.30  261  15  117  13  1.30  1.02, 1.67  1.27  0.98, 1.64  1.28  0.99, 1.65  1.19  0.91, 1.55  1.02  0.77, 1.34  Continuous          1.01  1.00, 1.02  1.00  0.99, 1.02  1.01  0.99, 1.02  1.00  0.99, 1.01  1.00  0.99, 1.01  Per 0.7-mg/L increase          1.04  0.98, 1.10  1.03  0.97, 1.09  1.03  0.97, 1.09  1.02  0.96, 1.07  0.99  0.93, 1.05  Abbreviations: BMI, body mass index; CI, confidence interval; OR, odds ratio. a Model 1 adjusted for age and sex. b Model 2 adjusted for age, sex, BMI, and smoking. c Model 3 adjusted for age, sex, BMI, smoking, and statin use. d Model 4 adjusted for age, sex, BMI, smoking, statin use, and cardiovascular disease. e Model 5 adjusted for age, sex, BMI, smoking, statin use, cardiovascular disease, and sports activity. Table 4. Risk of Venous Thrombosis According to Categories of Homocysteine Concentrations in Patients With Partners and Partner Controls, Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis, the Netherlands, 1999–2004 Homocysteine Concentration, mg/L  Patients With Partners  Partner Controls  Model 1a  Model 2b  Model 3c  Model 4d  Model 5e  No.  %  No.  %  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  <1.60  443  55  449  57  1.0  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.60–1.79  106  13  117  15  0.96  0.71, 1.29  0.95  0.70, 1.3  0.95  0.69, 1.30  1.05  0.76, 1.46  1.01  0.70, 1.44  1.80–2.09  80  10  76  10  1.10  0.77, 1.57  1.10  0.76, 1.61  1.09  0.75, 1.59  1.13  0.76, 1.70  1.33  0.86, 2.06  2.10–2.29  52  7  52  6  1.10  0.72, 1.68  1.13  0.73, 1.75  1.09  0.70, 1.70  1.24  0.77, 1.97  1.16  0.70, 1.94  ≥2.30  116  15  93  12  1.36  0.97, 1.92  1.40  0.97, 2.01  1.38  0.96, 1.99  1.38  0.94, 2.02  1.44  0.95, 2.17  Continuous          1.01  0.99, 1.02  1.01  0.99, 1.03  1.01  0.99, 1.03  1.01  0.99, 1.03  1.01  0.99, 1.03  Per 0.7-mg/L          1.05  0.97, 1.13  1.05  0.97, 1.14  1.05  0.97, 1.14  1.05  0.97, 1.14  1.05  0.96, 1.15  Homocysteine Concentration, mg/L  Patients With Partners  Partner Controls  Model 1a  Model 2b  Model 3c  Model 4d  Model 5e  No.  %  No.  %  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  <1.60  443  55  449  57  1.0  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.60–1.79  106  13  117  15  0.96  0.71, 1.29  0.95  0.70, 1.3  0.95  0.69, 1.30  1.05  0.76, 1.46  1.01  0.70, 1.44  1.80–2.09  80  10  76  10  1.10  0.77, 1.57  1.10  0.76, 1.61  1.09  0.75, 1.59  1.13  0.76, 1.70  1.33  0.86, 2.06  2.10–2.29  52  7  52  6  1.10  0.72, 1.68  1.13  0.73, 1.75  1.09  0.70, 1.70  1.24  0.77, 1.97  1.16  0.70, 1.94  ≥2.30  116  15  93  12  1.36  0.97, 1.92  1.40  0.97, 2.01  1.38  0.96, 1.99  1.38  0.94, 2.02  1.44  0.95, 2.17  Continuous          1.01  0.99, 1.02  1.01  0.99, 1.03  1.01  0.99, 1.03  1.01  0.99, 1.03  1.01  0.99, 1.03  Per 0.7-mg/L          1.05  0.97, 1.13  1.05  0.97, 1.14  1.05  0.97, 1.14  1.05  0.97, 1.14  1.05  0.96, 1.15  Abbreviations: BMI, body mass index; CI, confidence interval; OR, odds ratio. a Model 1 adjusted for age and sex, and partnership. b Model 2 adjusted for age, sex, BMI, smoking, and partnership. c Model 3 adjusted for age, sex, BMI, smoking, statin use, and partnership. d Model 4 adjusted for age, sex, BMI, smoking, statin use, cardiovascular disease, and partnership. e Model 5 adjusted for age, sex, BMI, smoking, statin use, cardiovascular disease, sports activity, and partnership. Table 4. Risk of Venous Thrombosis According to Categories of Homocysteine Concentrations in Patients With Partners and Partner Controls, Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis, the Netherlands, 1999–2004 Homocysteine Concentration, mg/L  Patients With Partners  Partner Controls  Model 1a  Model 2b  Model 3c  Model 4d  Model 5e  No.  %  No.  %  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  <1.60  443  55  449  57  1.0  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.60–1.79  106  13  117  15  0.96  0.71, 1.29  0.95  0.70, 1.3  0.95  0.69, 1.30  1.05  0.76, 1.46  1.01  0.70, 1.44  1.80–2.09  80  10  76  10  1.10  0.77, 1.57  1.10  0.76, 1.61  1.09  0.75, 1.59  1.13  0.76, 1.70  1.33  0.86, 2.06  2.10–2.29  52  7  52  6  1.10  0.72, 1.68  1.13  0.73, 1.75  1.09  0.70, 1.70  1.24  0.77, 1.97  1.16  0.70, 1.94  ≥2.30  116  15  93  12  1.36  0.97, 1.92  1.40  0.97, 2.01  1.38  0.96, 1.99  1.38  0.94, 2.02  1.44  0.95, 2.17  Continuous          1.01  0.99, 1.02  1.01  0.99, 1.03  1.01  0.99, 1.03  1.01  0.99, 1.03  1.01  0.99, 1.03  Per 0.7-mg/L          1.05  0.97, 1.13  1.05  0.97, 1.14  1.05  0.97, 1.14  1.05  0.97, 1.14  1.05  0.96, 1.15  Homocysteine Concentration, mg/L  Patients With Partners  Partner Controls  Model 1a  Model 2b  Model 3c  Model 4d  Model 5e  No.  %  No.  %  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  <1.60  443  55  449  57  1.0  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.60–1.79  106  13  117  15  0.96  0.71, 1.29  0.95  0.70, 1.3  0.95  0.69, 1.30  1.05  0.76, 1.46  1.01  0.70, 1.44  1.80–2.09  80  10  76  10  1.10  0.77, 1.57  1.10  0.76, 1.61  1.09  0.75, 1.59  1.13  0.76, 1.70  1.33  0.86, 2.06  2.10–2.29  52  7  52  6  1.10  0.72, 1.68  1.13  0.73, 1.75  1.09  0.70, 1.70  1.24  0.77, 1.97  1.16  0.70, 1.94  ≥2.30  116  15  93  12  1.36  0.97, 1.92  1.40  0.97, 2.01  1.38  0.96, 1.99  1.38  0.94, 2.02  1.44  0.95, 2.17  Continuous          1.01  0.99, 1.02  1.01  0.99, 1.03  1.01  0.99, 1.03  1.01  0.99, 1.03  1.01  0.99, 1.03  Per 0.7-mg/L          1.05  0.97, 1.13  1.05  0.97, 1.14  1.05  0.97, 1.14  1.05  0.97, 1.14  1.05  0.96, 1.15  Abbreviations: BMI, body mass index; CI, confidence interval; OR, odds ratio. a Model 1 adjusted for age and sex, and partnership. b Model 2 adjusted for age, sex, BMI, smoking, and partnership. c Model 3 adjusted for age, sex, BMI, smoking, statin use, and partnership. d Model 4 adjusted for age, sex, BMI, smoking, statin use, cardiovascular disease, and partnership. e Model 5 adjusted for age, sex, BMI, smoking, statin use, cardiovascular disease, sports activity, and partnership. Table 5. Risk of Venous Thrombosis in Men and Women According to Categories of Homocysteine Concentration, Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis, the Netherlands, 1999–2004 Homocysteine Concentration, mg/L  All Patients  RDD Controls  Adjusted for Age and Sex  Adjusted for Age, Sex, and Other Factorsa  No.  %  No.  %  OR  95% CI  OR  95% CI  Men   <1.60  370  44  241  49  1.00  Referent  1.00  Referent   1.60–1.79  143  17  89  18  1.01  0.74, 1.39  1.01  0.71, 1.44   1.80–2.09  123  14  57  12  1.36  0.96, 1.95  1.34  0.89, 2.03   2.10–2.29  58  7  28  6  1.25  0.77, 2.03  1.13  0.66, 1.93   ≥2.30  156  18  76  15  1.26  0.92, 1.74  1.08  0.75, 1.57  Women   <1.60  518  62  227  62  1.00  Referent  1.00  Referent   1.60–1.79  107  13  65  14  0.89  0.63, 1.25  0.75  0.48, 1.15   1.80–2.09  64  8  37  9  0.88  0.58, 1.35  0.76  0.45, 1.29   2.10–2.29  45  5  26  6  0.95  0.57, 1.58  0.77  0.42, 1.43   ≥2.30  105  12  41  9  1.41  0.95, 2.08  0.96  0.59, 1.54  Homocysteine Concentration, mg/L  All Patients  RDD Controls  Adjusted for Age and Sex  Adjusted for Age, Sex, and Other Factorsa  No.  %  No.  %  OR  95% CI  OR  95% CI  Men   <1.60  370  44  241  49  1.00  Referent  1.00  Referent   1.60–1.79  143  17  89  18  1.01  0.74, 1.39  1.01  0.71, 1.44   1.80–2.09  123  14  57  12  1.36  0.96, 1.95  1.34  0.89, 2.03   2.10–2.29  58  7  28  6  1.25  0.77, 2.03  1.13  0.66, 1.93   ≥2.30  156  18  76  15  1.26  0.92, 1.74  1.08  0.75, 1.57  Women   <1.60  518  62  227  62  1.00  Referent  1.00  Referent   1.60–1.79  107  13  65  14  0.89  0.63, 1.25  0.75  0.48, 1.15   1.80–2.09  64  8  37  9  0.88  0.58, 1.35  0.76  0.45, 1.29   2.10–2.29  45  5  26  6  0.95  0.57, 1.58  0.77  0.42, 1.43   ≥2.30  105  12  41  9  1.41  0.95, 2.08  0.96  0.59, 1.54  Abbreviations: CI, confidence interval; OR, odds ratio; RDD, random-digit dialing. a Additionally adjusted for body mass index, smoking, statin use, cardiovascular disease, sports activity, and (in women) hormone use. Table 5. Risk of Venous Thrombosis in Men and Women According to Categories of Homocysteine Concentration, Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis, the Netherlands, 1999–2004 Homocysteine Concentration, mg/L  All Patients  RDD Controls  Adjusted for Age and Sex  Adjusted for Age, Sex, and Other Factorsa  No.  %  No.  %  OR  95% CI  OR  95% CI  Men   <1.60  370  44  241  49  1.00  Referent  1.00  Referent   1.60–1.79  143  17  89  18  1.01  0.74, 1.39  1.01  0.71, 1.44   1.80–2.09  123  14  57  12  1.36  0.96, 1.95  1.34  0.89, 2.03   2.10–2.29  58  7  28  6  1.25  0.77, 2.03  1.13  0.66, 1.93   ≥2.30  156  18  76  15  1.26  0.92, 1.74  1.08  0.75, 1.57  Women   <1.60  518  62  227  62  1.00  Referent  1.00  Referent   1.60–1.79  107  13  65  14  0.89  0.63, 1.25  0.75  0.48, 1.15   1.80–2.09  64  8  37  9  0.88  0.58, 1.35  0.76  0.45, 1.29   2.10–2.29  45  5  26  6  0.95  0.57, 1.58  0.77  0.42, 1.43   ≥2.30  105  12  41  9  1.41  0.95, 2.08  0.96  0.59, 1.54  Homocysteine Concentration, mg/L  All Patients  RDD Controls  Adjusted for Age and Sex  Adjusted for Age, Sex, and Other Factorsa  No.  %  No.  %  OR  95% CI  OR  95% CI  Men   <1.60  370  44  241  49  1.00  Referent  1.00  Referent   1.60–1.79  143  17  89  18  1.01  0.74, 1.39  1.01  0.71, 1.44   1.80–2.09  123  14  57  12  1.36  0.96, 1.95  1.34  0.89, 2.03   2.10–2.29  58  7  28  6  1.25  0.77, 2.03  1.13  0.66, 1.93   ≥2.30  156  18  76  15  1.26  0.92, 1.74  1.08  0.75, 1.57  Women   <1.60  518  62  227  62  1.00  Referent  1.00  Referent   1.60–1.79  107  13  65  14  0.89  0.63, 1.25  0.75  0.48, 1.15   1.80–2.09  64  8  37  9  0.88  0.58, 1.35  0.76  0.45, 1.29   2.10–2.29  45  5  26  6  0.95  0.57, 1.58  0.77  0.42, 1.43   ≥2.30  105  12  41  9  1.41  0.95, 2.08  0.96  0.59, 1.54  Abbreviations: CI, confidence interval; OR, odds ratio; RDD, random-digit dialing. a Additionally adjusted for body mass index, smoking, statin use, cardiovascular disease, sports activity, and (in women) hormone use. The risk estimates for extremely high concentrations of homocysteine (in mg/L: 2.30–3.19, 3.20–6.40, and >6.40) are presented in Web Table 5. Crude and fully adjusted odds ratios for VT yielded null associations in all homocysteine categories, both for analyses with RDD controls and with partner controls. The highest (fully adjusted) odds ratio we found was in the matched analysis between patients and partner controls in the category of homocysteine concentrations between 3.2 and 6.4 mg/L, and it was 1.83 (95% CI: 0.95, 3.51). We investigated the association of homocysteine concentrations for subgroups of patients, restricting the analysis to provoked or unprovoked VT and DVT or pulmonary embolism (Web Table 6). A weak association was found between homocysteine concentrations higher than 2.3 mg/L and provoked VT as well as with DVT (age and sex–adjusted odds ratios were 1.37 (95% CI: 1.03, 1.83) and 1.34 (95% CI: 1.04, 1.74), respectively) when comparing patients with RDD controls. However, these findings were attenuated after adjustment for multiple confounding factors (for the provoked VT group, odds ratio = 1.08, 95% CI: 0.77, 1.50; for the DVT group, odds ratio = 1.15, 95% CI: 0.85, 1.56). Other odds ratios from this analysis were close to unity. DISCUSSION We analyzed data from the MEGA case-control study to investigate the association among different homocysteine concentrations and VT risk. We used 2 different control groups to study this association, population-derived controls identified using RDD and patients’ partner controls. Results from these 2 separate analyses demonstrated that hyperhomocysteinemia is not associated with increased risk of VT. The results were consistent when we used plasma homocysteine concentrations as a continuous variable and in terms of 0.7-mg/L increases. Any potential dose-response or threshold outcome was excluded. Results were consistent in men, women, patients with unprovoked or provoked VT, and in patients who had DVT or pulmonary embolism as the initial event. Our findings contradicted the results from 3 previous meta-analyses that reported hyperhomocysteinemia to be a risk factor for VT (4–6). The most recent meta-analysis included 3,289 patients and 3,780 controls from retrospective follow-up studies and 476 patients and 1,517 controls from prospective follow-up studies. The risks of VT per 0.7-mg/L (5 μmol/L) increase in plasma homocysteine were 1.60 (95% CI: 1.10, 2.34) and 1.27 (95% CI: 1.01, 1.59), respectively (4). However, the major problem of these meta-analyses is the possibility of confounding effects and publication bias. Our study included 1,689 patients and 1,726 controls. Moreover, we were able to adjust for many confounding factors not included in other studies due to small sample sizes. Additionally, we were able to exclude an association among homocysteine concentrations and DVT, pulmonary embolism, provoked VT, and unprovoked VT in the subgroup analyses. Our findings are in line with previous publication from the MEGA study, which reported no association between MTHFR genotype and VT (22). Because the MTHFR 677C→T variant has a phenotype in which homocysteine concentration is genetically increased, this finding provides further evidence that the association observed in previous studies cannot be causal. Two studies found sex differences in the association. The second Norwegian Health Study of Nord-Trøndelag (HUNT2) found that elevated homocysteine concentrations in men increased the frequency of a subsequent first VT 2-fold, whereas in women there was no relationship (8). In contrast, the Longitudinal Investigation of Thromboembolism Etiology (LITE) study, found that the association was higher in women (23). In MEGA, we did not find an association with VT in any of the postulated homocysteine concentrations when we analyzed men and women separately. Our null findings and understanding of the role of homocysteine in venous thrombotic disease are in line with the evolution of evidence for homocysteine as a potential target to modify arterial vascular disease risk. There too a promising hypothesis was first sparked by small retrospective studies in the 1990s (that is, a 2-fold increased risk of cardiovascular disease when homocysteine concentration was elevated) but returned to the sobering conclusion in 2012 (no increased risk) when all the evidence from published and unpublished studies was meta-analyzed with individual patient data and performing multiple adjustment for confounding factors (24). Although the MEGA study included a large number of individuals, we could not estimate in detail the risk of VT when the concentration of homocysteine was higher than 6.4 mg/L (i.e., concentrations that are associated with homocystinuria) given that n values were small (n = 16 patients, n = 7 RDD controls, and n = 6 partners). However, in one collaborative study, in which 5 centers from Ireland, Australia, the Netherlands, and the United States participated, information from patients with homocystinuria due to cystathionine β-synthase deficiency was registered, and a 90% reduction in vascular events was found after B-vitamin supplementation during an average treatment time of 17.9 years per patient (25). Patients with this diagnosis are expected to have plasma homocysteine concentrations above 6.4 mg/L, usually in the range of 13–65 mg/L (26). In terms of clinical implications, the present study, together with a previous study from MEGA on B-vitamin supplementation and VT risk (12), provides no evidence that B-vitamin supplementation will decrease VT risk in patients with plasma homocysteine concentrations below 6.4 mg/L without a diagnosis of homozygous cystathionine β-synthase deficiency. Certain limitations in this study should be noted. First, we excluded participants who were using B-vitamin supplementation, because vitamin-B intake lowers homocysteine concentrations (10, 11). Therefore, our results apply only to individuals who are not using B-vitamin therapy. Second, blood samples were collected in the outpatient setting, which could lead to an underrepresentation of chronically ill and bedridden patients. Third, our study was performed in individuals aged 18–70 years in a population that is mainly white in origin. Our results may be generalizable only to those who are well enough to visit an outpatient clinic and might not apply to the elderly and to other ethnicities. Fourth, there was no repeated or confirmatory capture of self-reported variables or measurement of fasting blood values, which might have led to misclassification in the study. Fifth, although our study is, to our knowledge, the largest case-control study on this issue to date, numbers in some subgroups were small, leading to large confidence intervals. Sixth, the MEGA study is a case-control study in which homocysteine concentrations were measured after the event. Whether these homocysteine concentrations are representative of levels before the event cannot be said with certainty. A previous meta-analysis found that of the 32 studies published at the time, 29 studies had homocysteine measurements from after the venous thrombotic event and only 3 studies had homocysteine measurements from prior to the event (4). The same meta-analysis showed that the association was stronger in the pooled retrospective studies than in the pooled prospective studies, and although this can be explained by several factors (such as regression dilution bias (27), an underestimation of the strength of the association as a certain exposure value at the beginning of follow-up might have changed at the time of event onset), reverse causation is likely to have played a role here. The fact that we found no association, in contrast to the previous retrospective studies, suggests that reverse causation was not present in our study. This may have been a result of 2 measures we took in the design of the study: 1) Measure plasma homocysteine concentration at least 3 months after VT occurred (we assume that an acute effect of VT leading to an increase in homocysteine concentration must have worn off); 2) exclude all pregnant women (who are folic acid users) and all individuals who reported B-vitamin supplementation at the time of blood draw (because B vitamins can lower homocysteine levels) from the study. Seventh, choosing controls in case-control studies can be difficult and can influence results. In the MEGA study, the partner controls may have been too closely matched to patients and could therefore yield null results, although conditional logistic regression should take this into account. In addition, RDD controls may be too healthy and therefore yield spurious results, given that health is related to lower homocysteine concentrations. A strength of the MEGA study is that both types of controls were included and that results from the study were independent of the type of control in our analyses. Eighth, because this is a matched case-control study, results could be contingent based on the single match that was made. The main message from this study is that when performing an observational study on the association of a certain biomarker with a specific disease, it is important to consider the drug therapy that can modify the biomarker concentration and other confounding factors that can bias the results. Building on this logic, the present observational study yielded the same results as randomized clinical trials that balance measured and unmeasured confounders. This study confirms what randomized clinical trials have shown: no association between homocysteine-lowering therapy and risk of VT (10, 11). In conclusion, in this study there was no evidence of an association among different homocysteine concentrations and increased risks for VT, whether for DVT, pulmonary embolism, provoked or unprovoked VT, or in men or women. ACKNOWLEDGMENTS Author affiliations: Department of Clinical Epidemiology, Leiden University Medical Center, Leiden, the Netherlands (Monica Ospina-Romero, Suzanne C. Cannegieter, Martin den Heijer, Carine J. M. Doggen, Frits. R. Rosendaal, Willem M. Lijfering); Department of Epidemiology and Biostatistics, University of California San Francisco, San Francisco, California (Monica Ospina-Romero); Department of Thrombosis and Hemostasis, Leiden University Medical Center, Leiden, the Netherlands (Suzanne C. Cannegieter); Department of Internal Medicine, VU Medical Center, Amsterdam, the Netherlands (Martin den Heijer); Department of Health Technology and Services Research, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, the Netherlands (Carine J. M. Doggen); and Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands (Frits. R. Rosendaal, Willem M. Lijfering). This research was supported by the Netherlands Heart Foundation (grant NHS 98.113), the Dutch Cancer Foundation (grant RUL 99/1992), and the Netherlands Organization for Scientific Research (grant 912-03-033 2003). The Netherlands Heart Foundation, the Dutch Cancer Foundation, and the Netherlands Organization for Scientific Research had no role in the design and conduct of the study; the collection, management, analysis, and interpretation of the data; or the preparation, review, or approval of the manuscript. Conflict of interest: none declared. Abbreviations CI confidence interval DVT deep vein thrombosis MEGA Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis RDD random digit-dialing VT venous thrombosis REFERENCES 1 ISTH Steering Committee for World Thrombosis Day. Thrombosis: a major contributor to the global disease burden. J Thromb Haemost . 2014; 12( 10): 1580– 1590. CrossRef Search ADS PubMed  2 Ray JG. Hyperhomocysteinemia: no longer a consideration in the management of venous thromboembolism. Curr Opin Pulm Med . 2008; 14( 5): 369– 373 Google Scholar CrossRef Search ADS PubMed  3 McCully KS. Vascular pathology of homocysteinemia: implications for the pathogenesis of arteriosclerosis. Am J Pathol . 1969; 56( 1): 111– 128 Google Scholar PubMed  4 den Heijer M, Lewington S, Clarke R. Homocysteine, MTHFR and risk of venous thrombosis: a meta-analysis of published epidemiological studies. J Thromb Haemost . 2005; 3( 2): 292– 299. 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Google Scholar CrossRef Search ADS PubMed  13 Loscalzo J. Homocysteine trials—clear outcomes for complex reasons. N Engl J Med . 2006; 354( 15): 1629– 1632. Google Scholar CrossRef Search ADS PubMed  14 Pomp ER, Van Stralen KJ, Le Cessie S, et al.  . Experience with multiple control groups in a large population-based case-control study on genetic and environmental risk factors. Eur J Epidemiol . 2010; 25( 7): 459– 466. Google Scholar CrossRef Search ADS PubMed  15 Willems HP, Bos GM, Gerrits WB, et al.  . Acidic citrate stabilizes blood samples for assay of total homocysteine. Clin Chem . 1998; 44( 2): 342– 345. Google Scholar PubMed  16 Van Allen MI, Fraser FC, Dallaire L, et al.  . Recommendations on the use of folic acid supplementation to prevent the recurrence of neural tube defects. Clinical Teratology Committee, Canadian College of Medical Geneticists. CMAJ . 1993; 149( 9): 1239– 1243. Google Scholar PubMed  17 den Heijer M, Koster T, Blom HJ, et al.  . Hyperhomocysteinemia as a risk factor for deep-vein thrombosis. N Engl J Med . 1996; 334( 12): 759– 762. Google Scholar CrossRef Search ADS PubMed  18 Roach RE, Lijfering WM, Rosendaal FR, et al.  . Sex difference in risk of second but not of first venous thrombosis: paradox explained. Circulation . 2014; 129( 1): 51– 56 Google Scholar CrossRef Search ADS PubMed  19 Rozemeijer K, le Cessie S, van Hylckama Vlieg A, et al.  . Exposure opportunity: the advantages of including men in analyses of female-related risk factors. Am J Epidemiol . 2017; 185( 10): 965– 973. Google Scholar CrossRef Search ADS PubMed  20 Breslow NE, Day NE. Statistical Methods in Cancer Research . Lyon, France: International Agency for Research on Cancer; 1980. 21 IBM Knowledge Center. SPSS statistics 21.0.0. Logistic Regression. https://www.ibm.com/support/knowledgecenter/en/SSLVMB_21.0.0/com.ibm.spss.statistics.help/syn_logistic_regression.htm. Accessed January 3, 2018. 22 Bezemer ID, Doggen CJ, Vos HL, et al.  . No association between the common MTHFR 677C->T polymorphism and venous thrombosis: results from the MEGA study. Arch Intern Med . 2007; 167( 5): 497– 501. Google Scholar CrossRef Search ADS PubMed  23 Tsai AW, Cushman M, Tsai MY, et al.  . Serum homocysteine, thermolabile variant of methylene tetrahydrofolate reductase (MTHFR), and venous thromboembolism: Longitudinal Investigation of Thromboembolism Etiology (LITE) [published erratum appears in Am J Hematol. 2007;82(11):1031–1032]. Am J Hematol . 2003; 72( 3): 192– 200. Google Scholar CrossRef Search ADS PubMed  24 Lewington S, Bragg F, Clarke R. A review on metaanalysis of biomarkers: promises and pitfalls. Clin Chem . 2012; 58( 8): 1192– 1204. Google Scholar CrossRef Search ADS PubMed  25 Yap S, Boers GH, Wilcken B, et al.  . Vascular outcome in patients with homocystinuria due to cystathionine beta-synthase deficiency treated chronically: a multicenter observational study. Arterioscler Thromb Vasc Biol . 2001; 21( 12): 2080– 2085. Google Scholar CrossRef Search ADS PubMed  26 Eldibany MM, Caprini JA. Hyperhomocysteinemia and thrombosis: an overview. Arch Pathol Lab Med . 2007; 131( 6): 872– 884. Google Scholar PubMed  27 Clarke R, Shipley M, Lewington S, et al.  . Underestimation of risk associations due to regression dilution in long-term follow-up of prospective studies. Am J Epidemiol . 1999; 150( 4): 341– 353. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png American Journal of Epidemiology Oxford University Press

Hyperhomocysteinemia and Risk of First Venous Thrombosis: The Influence of (Unmeasured) Confounding Factors

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Abstract

Abstract Meta-analyses have reported a 2- to 3-fold increased risk of venous thrombosis (VT) in individuals with hyperhomocysteinemia. However, confounding factors were generally not considered. In contrast, randomized trials of homocysteine-lowering therapy and VT risk have been negative. We investigated whether hyperhomocysteinemia was associated with VT in the Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis (MEGA) case-control study (1999–2004) from the Netherlands (1,689 cases and 1,726 controls), taking into account measured and unmeasured confounders. We compared patients with population controls to estimate odds ratios using unconditional logistic regression and adjusted for various potential confounders. We matched patients to their partners to additionally adjust for unmeasured confounders (e.g., lifestyle factors) using conditional logistic regression. We found that elevated homocysteine concentrations were not associated with an increased risk for VT when comparing patients with population controls, either as a continuous variable (odds ratio = 1.00, 95% confidence interval: 0.99, 1.01), in terms of 0.7-mg/L increase (odds ratio = 0.99, 95% confidence interval: 0.93, 1.05), or within different homocysteine categories. We obtained similar results when patients were compared with their partners. Stratification by sex, deep vein thrombosis, pulmonary embolism, provoked VT, and unprovoked VT also provided no evidence of an association. In conclusion, after extensive adjustments for confounding, hyperhomocysteinemia was not associated with an increased risk of venous thrombosis in this study. homocysteine, homocystinuria, venous thrombosis, vitamin supplementation Venous thrombosis (VT) accounts for an important burden of disease in the world (1). A systematic review of the literature reported an overall annual incidence of VT ranging from 0.75 to 2.69 per 1,000 individuals in Western Europe, North America, Australia, and Latin America (1). Multiple risk factors for VT have been reported; one is hyperhomocysteinemia, but its causal association with VT has remained controversial (2). Homocysteine is an intermediate amino acid in the metabolism of methionine and cysteine. It was first linked to cardiovascular disease and VT in 1969 (3). Three meta-analyses demonstrated a modest association of increased VT risk per 0.7-mg/L (5 μmol/L) higher homocysteine concentration or hyperhomocysteinemia (4–6). However, the available evidence makes it difficult to discern whether the association between hyperhomocysteinemia and VT is a causal or spurious phenomenon. First, proper adjustment for age, sex, and many lifestyle-related factors that could confound the relationship was not feasible in the meta-analyses because most studies did not report an adjusted odds ratio due to small sample sizes. For instance, in the 1990s, case-control studies usually included no more than 75 cases with VT with similarly few controls (4). The numbers of cases and controls since then have also been fairly small, with a maximum number of 397 cases with VT and 585 controls in a study from 2001 (7). Second, previous studies defined hyperhomocysteinemia differently (i.e., homocysteine concentrations above the 95th percentile or mean plus 2 standard deviations calculated from the distribution in control groups), and the cutoff levels for presence of hyperhomocysteinemia, when mentioned, ranged from >1.1 mg/L to >3.4 mg/L (4, 8). Third, these meta-analyses did not exclude publication bias, which could have resulted in an overestimation of the risk. And fourth, some studies have suggested that the association of homocysteine with VT is visible only in subgroups in which conventional risk factors for VT are absent (9). Hyperhomocysteinemia can be treated with B-vitamin supplementation (folic acid, pyridoxine, and cobalamin), but several studies (including 2 clinical trials) failed to demonstrate any benefit from such homocysteine-lowering therapy in the prevention of VT (10–12). It has been argued that the biological mechanism through which homocysteine increases the risk of vascular disease is more complex than an increase in homocysteine only (13). Additionally, these clinical trials present some limitations that preclude the conclusion that the reported association between homocysteine and VT was related to the effect of confounders. For example, the follow-up time (average of less than 4 years) might have been too short to show a risk reduction in VT incidence among individuals treated with homocysteine-lowering treatment. In addition, the generalizability of results might not be applicable to the general population, because the 2 clinical trials that evaluated the benefit of homocysteine-lowering therapy were conducted either among patients with a prior history of VT or among those with a higher risk of cardiovascular disease (10, 11). We decided to report on the association among various concentrations of homocysteine and VT in a single, large, population-based case-control study to examine whether the association between hyperhomocysteinemia and first VT event persisted after adjustment for both measured and unmeasured confounding factors. The analysis included 1,689 patients with a first VT event, either deep vein thrombosis (DVT) of the leg or pulmonary embolism, and 1,726 control subjects from the Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis (MEGA) study. This study enabled us to compare associations with VT for various homocysteine cutoff levels in various subgroups in which we could adjust for both measured and unmeasured confounding factors by comparing patients with population-derived (using random-digit dialing (RDD)) controls and with the patients’ partners, which formed the 2 control groups of the study. METHODS Study population The design of the MEGA case-control study is described elsewhere (14). In short, MEGA is a large, population-based case-control study that recruited 4,956 cases with a first diagnosis of deep VT or pulmonary embolism, from 6 anticoagulation clinics in the Netherlands, between March 1999 and September 2004. The diagnosis of DVT was confirmed by Doppler ultrasonography. The diagnosis of pulmonary embolism was made by ventilation-perfusion lung scan, spiral computed tomography, or angiogram. Two different control groups were invited to participate, patients’ partners (n = 3,295) and controls identified using RDD (n = 3,000). The latter control group was recruited from January 2002 through September 2004. These participants were also between 18 and 70 years of age, with no previous history of VT. For logistical reasons, blood sampling was performed for participants included up to June 2002. Data collection and definitions The date of diagnosis was assigned as the index date for patients and their partner controls, and for RDD controls, the index date was the date of signing informed consent. All participants were asked to complete a questionnaire about demographic characteristics, lifestyle, risk factors for VT, and cardiovascular diseases. Body mass index was calculated using self-reported weight and height. Smoking habits were classified as current smoker, previous smoker, or nonsmoker. Risk factors for provoked VT (prior surgery, plaster cast immobilization, trauma, hospitalization in the previous 3 months, long-distance travel in the preceding 2 months, or malignancy at the time of or in the 5 years before the index date) were obtained with the questionnaire. Women were also asked about the use of hormone therapy (hormonal replacement therapy or oral-contraceptive use). Participants were asked about cardiovascular disease (self-reported previous myocardial infarction or ischemic stroke) and statin use. Individuals involved in sports activities at least once a week were considered to be practicing regular sports activity. Laboratory measurements Fasting blood samples were taken from patients approximately 3 months after ending anticoagulation therapy. In patients who continued their anticoagulation therapy, blood was sampled 1 year after the index date. Partner controls provided their fasting blood sample along with their partners (patients). RDD controls were invited for a fasting blood draw after returning their questionnaires. Blood samples were collected in tubes with citrate anticoagulant to prevent increases in homocysteine concentration (15). Total homocysteine plasma concentrations were measured in a central laboratory (Laboratory of Pediatrics and Neurology in Nijmegen, the Netherlands) by an automated high-performance liquid chromatography method with reverse-phase and fluorescent detection (Gilson 232-401 sample processor (Gilson Inc., Middleton, Wisconsin), Spectra Physics 8800 solvent delivery system, and Spectra Physics LC 304 fluorometer (Spectra-Physics, San Jose, California)). Inclusion and exclusion criteria Of the 4,956 patients, we excluded 182 women who were pregnant at the index date or within the previous 3 months. These women were excluded because guidelines recommend folic acid during pregnancy and pregnancy itself affects risk (16). Next, we excluded 1,467 patients whose vitamin consumption information was missing or who were using vitamin-B therapy, because trials have shown that vitamin B intake lowers plasma homocysteine concentrations without affecting VT risk (10, 11). Finally, we excluded 1,618 patients who were included in the study after June 2002 because blood samples were no longer drawn from this date onward. This left us with 1,689 patients. Of these patients, 787 had a partner who fulfilled the inclusion criteria and was willing to participate, so 787 matched pairs remained. After application of the same exclusion criteria on the RDD control group, 939 RDD control participants were included in the analysis. Statistical analysis The analysis of plasma homocysteine concentrations was carried out in 2 separate ways, as a continuous (per 0.13-mg/L increase or 1-μmol/L increase) variable and using categories. Plasma concentrations of homocysteine were divided into categories of 0.26 mg/L. Values lower than 1.6 mg/L were allocated to the lowest (reference) category, and values higher than 2.3 mg/L to the highest category. Next, we performed an analysis for extremely high versus normal concentrations of homocysteine using the following categories: in mg/L, 2.30–3.19, 3.20–6.49, and ≥6.50. We set 2.3 mg/L as the cutoff value because a previous analysis in the Leiden Thrombophilia Study (LETS) showed that VT risk was increased only for concentrations over 2.3 mg/L (18 μmol/L) (17). The analysis was repeated with 0.7-mg/L intervals because a previous meta-analysis observed a 1.3- to 1.6-fold increased risk per 0.7-mg/L (5 μmol/L) increase of homocysteine (4). Odds ratios with 95% confidence intervals were calculated as an estimate of the relative risk of VT for the different concentrations of homocysteine. When the analysis was made for all patients and RDD controls, we used unconditional logistic regression, adjusting for age and sex. Analyses were further adjusted for body mass index, smoking, statin use, history of arterial cardiovascular disease, and regular sports activities. Because some studies indicate a different relationship between elevated homocysteine concentration and VT in women and men (12, 18, 19), we additionally performed an analysis stratified by sex. Within this sex-stratified analysis, we additionally adjusted for hormone therapy in women. Because partners of patients are likely to resemble the patients in health behavior more than do random-digit dialing controls, we performed a 1:1-matched analysis by conditional logistic regression, which adjusts for associations within matched pairs. This method provides adjustment for all unmeasured factors for which couples tend to be similar (20). The analysis is conditional given that many clinical characteristics of controls, who are individually matched to the patients, are likely to be similar to patients’ characteristics. In this analysis, all aforementioned potential confounding factors were also adjusted for. Although using partners as controls results in most controls having the opposite sex as their matched case, one can adjust for sex in a partner-matched case-control study by allowing for sex with an indicator variable (19). Nevertheless, we also performed an analysis where only men were compared with men and women with women. Furthermore, odds ratios were calculated to estimate risk for VT associated with homocysteine concentrations of ≥2.3 mg/L, using as reference range homocysteine <2.3 mg/L for patients with provoked or unprovoked VT, and patients with deep vein thrombosis, pulmonary embolism, or both in subgroup analyses. All statistical analyses were performed using SPSS for Windows, release 20.0 (SPSS Inc., Chicago, Illinois). Conditional logistic regression was performed using the COXREG procedure (21). Because many studies on homocysteine report homocysteine concentrations in μmol/L instead of mg/L, we decided to show our analyses on homocysteine concentrations in both mg/L (in the main article) and in μmol/L (in Web Tables 1–6, available at https://academic.oup.com/aje) RESULTS A total of 3,415 participants (1,689 patients, 787 partner controls, and 939 RDD controls) were included in the study (Figure 1). The main characteristics of the participants are presented in Tables 1 and 2. The mean age was 49 years in patients, 50 years in partner controls, and 48 years in RDD controls (all in age range 18–70 years). Homocysteine concentrations above 2.3 mg/L were present in 261 (15%) patients, 93 (12%) partner controls, and 117 (13%) RDD controls. In general, in the category of homocysteine concentrations lower than 1.6 mg/L, more people of young age, female sex, or engaged in sports activities were present than in the other categories. As expected, VT risk factors were more often present in patients than in controls. Figure 1. View largeDownload slide Selection of patients with first venous thrombosis (VT) and controls in the Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis (MEGA) study, the Netherlands, 1999–2004. A) Selection of patients with history of VT; B) selection of partner controls; C) selection of controls identified using random-digit dialing (RDD). Figure 1. View largeDownload slide Selection of patients with first venous thrombosis (VT) and controls in the Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis (MEGA) study, the Netherlands, 1999–2004. A) Selection of patients with history of VT; B) selection of partner controls; C) selection of controls identified using random-digit dialing (RDD). Table 1. Clinical Characteristics of Patients With Venous Thrombosis Included in the Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis Case-Control Study, the Netherlands, 1999–2004 Characteristica  Homocysteine Concentration, mg/L  Overall  <1.60  1.60–1.79  1.80–2.09  2.10–2.29  ≥2.30  No.  %  No.  %  No.  %  No.  %  No.  %  No.  %  Total  1,689  888  250  187  103  261  Men  850  50  370  42  143  57  123  66  58  56  156  60  Age at enrollment, yearsb  49 (18–70)  47 (18–70)  50 (19–70)  50 (18–70)  52 (21–70)  52 (19–70)  BMIb,c  27 (17–58)  27 (17–58)  27 (18–47)  27 (18–46)  27 (18–40)  27 (18–49)  Malignancy  89  5  39  4  8  3  13  7  10  10  19  7  Hormone use (in women)  514  62  343  67  56  52  42  67  24  56  49  48  Risk factors for venous thrombosis                           Provokedd  863  51  497  56  107  43  91  49  51  50  117  45   Unprovoked  818  49  387  44  143  57  95  51  51  50  142  55  Arterial cardiovascular risk factors                           Previous smoking  520  31  263  30  76  31  62  33  29  28  90  35   Current smoking  584  35  278  31  92  37  68  37  41  40  105  40   Self-reported prior CVDe  55  4  15  2  13  6  10  6  3  3  14  6   Statin use  62  4  33  4  11  4  4  2  1  1  13  5   Regular sports activity  512  34  292  37  82  37  55  33  23  25  60  27  Characteristica  Homocysteine Concentration, mg/L  Overall  <1.60  1.60–1.79  1.80–2.09  2.10–2.29  ≥2.30  No.  %  No.  %  No.  %  No.  %  No.  %  No.  %  Total  1,689  888  250  187  103  261  Men  850  50  370  42  143  57  123  66  58  56  156  60  Age at enrollment, yearsb  49 (18–70)  47 (18–70)  50 (19–70)  50 (18–70)  52 (21–70)  52 (19–70)  BMIb,c  27 (17–58)  27 (17–58)  27 (18–47)  27 (18–46)  27 (18–40)  27 (18–49)  Malignancy  89  5  39  4  8  3  13  7  10  10  19  7  Hormone use (in women)  514  62  343  67  56  52  42  67  24  56  49  48  Risk factors for venous thrombosis                           Provokedd  863  51  497  56  107  43  91  49  51  50  117  45   Unprovoked  818  49  387  44  143  57  95  51  51  50  142  55  Arterial cardiovascular risk factors                           Previous smoking  520  31  263  30  76  31  62  33  29  28  90  35   Current smoking  584  35  278  31  92  37  68  37  41  40  105  40   Self-reported prior CVDe  55  4  15  2  13  6  10  6  3  3  14  6   Statin use  62  4  33  4  11  4  4  2  1  1  13  5   Regular sports activity  512  34  292  37  82  37  55  33  23  25  60  27  Abbreviations: BMI, body mass index; CVD, cardiovascular disease. a Data were missing for some participants in some subgroups. b Values are expressed as mean (range). c Weight (kg)/height (m2). d Provoked: previous surgery, malignancy, immobilization, trauma, plaster cast, oral contraceptive/hormonal replacement therapy, or recent travel. e CVD denotes self-reported myocardial infarction or ischemic stroke. View Large Table 1. Clinical Characteristics of Patients With Venous Thrombosis Included in the Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis Case-Control Study, the Netherlands, 1999–2004 Characteristica  Homocysteine Concentration, mg/L  Overall  <1.60  1.60–1.79  1.80–2.09  2.10–2.29  ≥2.30  No.  %  No.  %  No.  %  No.  %  No.  %  No.  %  Total  1,689  888  250  187  103  261  Men  850  50  370  42  143  57  123  66  58  56  156  60  Age at enrollment, yearsb  49 (18–70)  47 (18–70)  50 (19–70)  50 (18–70)  52 (21–70)  52 (19–70)  BMIb,c  27 (17–58)  27 (17–58)  27 (18–47)  27 (18–46)  27 (18–40)  27 (18–49)  Malignancy  89  5  39  4  8  3  13  7  10  10  19  7  Hormone use (in women)  514  62  343  67  56  52  42  67  24  56  49  48  Risk factors for venous thrombosis                           Provokedd  863  51  497  56  107  43  91  49  51  50  117  45   Unprovoked  818  49  387  44  143  57  95  51  51  50  142  55  Arterial cardiovascular risk factors                           Previous smoking  520  31  263  30  76  31  62  33  29  28  90  35   Current smoking  584  35  278  31  92  37  68  37  41  40  105  40   Self-reported prior CVDe  55  4  15  2  13  6  10  6  3  3  14  6   Statin use  62  4  33  4  11  4  4  2  1  1  13  5   Regular sports activity  512  34  292  37  82  37  55  33  23  25  60  27  Characteristica  Homocysteine Concentration, mg/L  Overall  <1.60  1.60–1.79  1.80–2.09  2.10–2.29  ≥2.30  No.  %  No.  %  No.  %  No.  %  No.  %  No.  %  Total  1,689  888  250  187  103  261  Men  850  50  370  42  143  57  123  66  58  56  156  60  Age at enrollment, yearsb  49 (18–70)  47 (18–70)  50 (19–70)  50 (18–70)  52 (21–70)  52 (19–70)  BMIb,c  27 (17–58)  27 (17–58)  27 (18–47)  27 (18–46)  27 (18–40)  27 (18–49)  Malignancy  89  5  39  4  8  3  13  7  10  10  19  7  Hormone use (in women)  514  62  343  67  56  52  42  67  24  56  49  48  Risk factors for venous thrombosis                           Provokedd  863  51  497  56  107  43  91  49  51  50  117  45   Unprovoked  818  49  387  44  143  57  95  51  51  50  142  55  Arterial cardiovascular risk factors                           Previous smoking  520  31  263  30  76  31  62  33  29  28  90  35   Current smoking  584  35  278  31  92  37  68  37  41  40  105  40   Self-reported prior CVDe  55  4  15  2  13  6  10  6  3  3  14  6   Statin use  62  4  33  4  11  4  4  2  1  1  13  5   Regular sports activity  512  34  292  37  82  37  55  33  23  25  60  27  Abbreviations: BMI, body mass index; CVD, cardiovascular disease. a Data were missing for some participants in some subgroups. b Values are expressed as mean (range). c Weight (kg)/height (m2). d Provoked: previous surgery, malignancy, immobilization, trauma, plaster cast, oral contraceptive/hormonal replacement therapy, or recent travel. e CVD denotes self-reported myocardial infarction or ischemic stroke. View Large Table 2. Clinical Characteristics of Controls Included in the Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis Case-Control Study, the Netherlands, 1999–2004 Characteristica  Homocysteine Concentration, mg/L  Overall  <1.60  1.60–1.79  1.80–2.09  2.10–2.29  ≥2.30  No.  %  No.  %  No.  %  No.  %  No.  %  No.  %  Partners of Patients  Total  787  449  117  76  52  93  Men  386  49  196  44  59  50  44  58  32  62  55  59  Age at enrollment, yearsb  50 (18–70)  49 (18–70)  51 (28–68)  50 (26–70)  54 (30–70)  50 (21–70)  BMIb,c  27 (17–52)  26 (17–44)  26 (19–48)  27 (20–42)  26 (19–40)  25 (18–45)  Malignancy  10  1  4  1  2  2  2  3  1  2  1  1  Hormone use (in women)  78  20  59  24  9  17  4  13  3  15  3  8  Risk factors for venous thrombosis                           Provokedd  112  14  78  18  14  13  8  11  6  12  6  7   Unprovoked  661  86  366  82  98  87  66  89  46  88  85  93  Arterial cardiovascular risk factors                           Previous smoking  241  31  140  31  39  34  22  29  13  26  27  29   Current smoking  230  29  125  28  28  24  25  33  17  33  35  38   Self-reported prior CVDe  18  3  10  2  2  2  1  2  1  2  4  5   Statin use  43  6  26  6  7  6  3  4  3  6  4  4   Regular sports activity  263  38  165  41  33  32  23  35  12  27  30  37  Controls Identified by Random-Digit Dialing  Total  939  518  154  96  54  117  Men  491  52  241  47  89  58  57  59  28  52  76  65  Age at enrollment, yearsb  48 (18–70)  47 (18–70)  49 (20–70)  50 (20–70)  52 (23–70)  50 (19–69)  BMIb,c  25 (16–43)  25 (16–40)  26 (19–43)  26 (17–39)  26 (20–39)  25 (18–37)  Malignancy  20  2  11  2  3  2  2  2  2  4  2  2  Hormone use (% in women)  145  33  98  36  18  29  9  23  5  19  15  37  Risk factors for venous thrombosis                           Provokedd  195  21  126  25  26  17  12  13  9  17  22  19   Unprovoked  733  79  386  75  125  83  83  87  45  83  94  81  Arterial cardiovascular risk factors                           Previous smoking  289  31  150  29  49  32  37  39  17  32  36  31   Current smoking  279  30  147  29  39  26  29  30  18  33  46  40   Self-reported prior CVDe  26  3  14  3  3  2  4  4  1  2  4  3   Statin use  79  8  40  8  13  8  10  10  3  6  13  11   Regular sports activity  396  48  233  52  68  49  36  44  22  44  37  34  Characteristica  Homocysteine Concentration, mg/L  Overall  <1.60  1.60–1.79  1.80–2.09  2.10–2.29  ≥2.30  No.  %  No.  %  No.  %  No.  %  No.  %  No.  %  Partners of Patients  Total  787  449  117  76  52  93  Men  386  49  196  44  59  50  44  58  32  62  55  59  Age at enrollment, yearsb  50 (18–70)  49 (18–70)  51 (28–68)  50 (26–70)  54 (30–70)  50 (21–70)  BMIb,c  27 (17–52)  26 (17–44)  26 (19–48)  27 (20–42)  26 (19–40)  25 (18–45)  Malignancy  10  1  4  1  2  2  2  3  1  2  1  1  Hormone use (in women)  78  20  59  24  9  17  4  13  3  15  3  8  Risk factors for venous thrombosis                           Provokedd  112  14  78  18  14  13  8  11  6  12  6  7   Unprovoked  661  86  366  82  98  87  66  89  46  88  85  93  Arterial cardiovascular risk factors                           Previous smoking  241  31  140  31  39  34  22  29  13  26  27  29   Current smoking  230  29  125  28  28  24  25  33  17  33  35  38   Self-reported prior CVDe  18  3  10  2  2  2  1  2  1  2  4  5   Statin use  43  6  26  6  7  6  3  4  3  6  4  4   Regular sports activity  263  38  165  41  33  32  23  35  12  27  30  37  Controls Identified by Random-Digit Dialing  Total  939  518  154  96  54  117  Men  491  52  241  47  89  58  57  59  28  52  76  65  Age at enrollment, yearsb  48 (18–70)  47 (18–70)  49 (20–70)  50 (20–70)  52 (23–70)  50 (19–69)  BMIb,c  25 (16–43)  25 (16–40)  26 (19–43)  26 (17–39)  26 (20–39)  25 (18–37)  Malignancy  20  2  11  2  3  2  2  2  2  4  2  2  Hormone use (% in women)  145  33  98  36  18  29  9  23  5  19  15  37  Risk factors for venous thrombosis                           Provokedd  195  21  126  25  26  17  12  13  9  17  22  19   Unprovoked  733  79  386  75  125  83  83  87  45  83  94  81  Arterial cardiovascular risk factors                           Previous smoking  289  31  150  29  49  32  37  39  17  32  36  31   Current smoking  279  30  147  29  39  26  29  30  18  33  46  40   Self-reported prior CVDe  26  3  14  3  3  2  4  4  1  2  4  3   Statin use  79  8  40  8  13  8  10  10  3  6  13  11   Regular sports activity  396  48  233  52  68  49  36  44  22  44  37  34  Abbreviations: BMI, body mass index; CVD, cardiovascular disease. a Data were missing for some participants in some subgroups. b Values are expressed as mean (range). c Weight (kg)/height (m2). d Provoked: previous surgery, malignancy, immobilization, trauma, plaster cast, oral contraceptive/hormonal replacement therapy, or recent travel. e CVD denotes self-reported myocardial infarction or ischemic stroke. View Large Table 2. Clinical Characteristics of Controls Included in the Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis Case-Control Study, the Netherlands, 1999–2004 Characteristica  Homocysteine Concentration, mg/L  Overall  <1.60  1.60–1.79  1.80–2.09  2.10–2.29  ≥2.30  No.  %  No.  %  No.  %  No.  %  No.  %  No.  %  Partners of Patients  Total  787  449  117  76  52  93  Men  386  49  196  44  59  50  44  58  32  62  55  59  Age at enrollment, yearsb  50 (18–70)  49 (18–70)  51 (28–68)  50 (26–70)  54 (30–70)  50 (21–70)  BMIb,c  27 (17–52)  26 (17–44)  26 (19–48)  27 (20–42)  26 (19–40)  25 (18–45)  Malignancy  10  1  4  1  2  2  2  3  1  2  1  1  Hormone use (in women)  78  20  59  24  9  17  4  13  3  15  3  8  Risk factors for venous thrombosis                           Provokedd  112  14  78  18  14  13  8  11  6  12  6  7   Unprovoked  661  86  366  82  98  87  66  89  46  88  85  93  Arterial cardiovascular risk factors                           Previous smoking  241  31  140  31  39  34  22  29  13  26  27  29   Current smoking  230  29  125  28  28  24  25  33  17  33  35  38   Self-reported prior CVDe  18  3  10  2  2  2  1  2  1  2  4  5   Statin use  43  6  26  6  7  6  3  4  3  6  4  4   Regular sports activity  263  38  165  41  33  32  23  35  12  27  30  37  Controls Identified by Random-Digit Dialing  Total  939  518  154  96  54  117  Men  491  52  241  47  89  58  57  59  28  52  76  65  Age at enrollment, yearsb  48 (18–70)  47 (18–70)  49 (20–70)  50 (20–70)  52 (23–70)  50 (19–69)  BMIb,c  25 (16–43)  25 (16–40)  26 (19–43)  26 (17–39)  26 (20–39)  25 (18–37)  Malignancy  20  2  11  2  3  2  2  2  2  4  2  2  Hormone use (% in women)  145  33  98  36  18  29  9  23  5  19  15  37  Risk factors for venous thrombosis                           Provokedd  195  21  126  25  26  17  12  13  9  17  22  19   Unprovoked  733  79  386  75  125  83  83  87  45  83  94  81  Arterial cardiovascular risk factors                           Previous smoking  289  31  150  29  49  32  37  39  17  32  36  31   Current smoking  279  30  147  29  39  26  29  30  18  33  46  40   Self-reported prior CVDe  26  3  14  3  3  2  4  4  1  2  4  3   Statin use  79  8  40  8  13  8  10  10  3  6  13  11   Regular sports activity  396  48  233  52  68  49  36  44  22  44  37  34  Characteristica  Homocysteine Concentration, mg/L  Overall  <1.60  1.60–1.79  1.80–2.09  2.10–2.29  ≥2.30  No.  %  No.  %  No.  %  No.  %  No.  %  No.  %  Partners of Patients  Total  787  449  117  76  52  93  Men  386  49  196  44  59  50  44  58  32  62  55  59  Age at enrollment, yearsb  50 (18–70)  49 (18–70)  51 (28–68)  50 (26–70)  54 (30–70)  50 (21–70)  BMIb,c  27 (17–52)  26 (17–44)  26 (19–48)  27 (20–42)  26 (19–40)  25 (18–45)  Malignancy  10  1  4  1  2  2  2  3  1  2  1  1  Hormone use (in women)  78  20  59  24  9  17  4  13  3  15  3  8  Risk factors for venous thrombosis                           Provokedd  112  14  78  18  14  13  8  11  6  12  6  7   Unprovoked  661  86  366  82  98  87  66  89  46  88  85  93  Arterial cardiovascular risk factors                           Previous smoking  241  31  140  31  39  34  22  29  13  26  27  29   Current smoking  230  29  125  28  28  24  25  33  17  33  35  38   Self-reported prior CVDe  18  3  10  2  2  2  1  2  1  2  4  5   Statin use  43  6  26  6  7  6  3  4  3  6  4  4   Regular sports activity  263  38  165  41  33  32  23  35  12  27  30  37  Controls Identified by Random-Digit Dialing  Total  939  518  154  96  54  117  Men  491  52  241  47  89  58  57  59  28  52  76  65  Age at enrollment, yearsb  48 (18–70)  47 (18–70)  49 (20–70)  50 (20–70)  52 (23–70)  50 (19–69)  BMIb,c  25 (16–43)  25 (16–40)  26 (19–43)  26 (17–39)  26 (20–39)  25 (18–37)  Malignancy  20  2  11  2  3  2  2  2  2  4  2  2  Hormone use (% in women)  145  33  98  36  18  29  9  23  5  19  15  37  Risk factors for venous thrombosis                           Provokedd  195  21  126  25  26  17  12  13  9  17  22  19   Unprovoked  733  79  386  75  125  83  83  87  45  83  94  81  Arterial cardiovascular risk factors                           Previous smoking  289  31  150  29  49  32  37  39  17  32  36  31   Current smoking  279  30  147  29  39  26  29  30  18  33  46  40   Self-reported prior CVDe  26  3  14  3  3  2  4  4  1  2  4  3   Statin use  79  8  40  8  13  8  10  10  3  6  13  11   Regular sports activity  396  48  233  52  68  49  36  44  22  44  37  34  Abbreviations: BMI, body mass index; CVD, cardiovascular disease. a Data were missing for some participants in some subgroups. b Values are expressed as mean (range). c Weight (kg)/height (m2). d Provoked: previous surgery, malignancy, immobilization, trauma, plaster cast, oral contraceptive/hormonal replacement therapy, or recent travel. e CVD denotes self-reported myocardial infarction or ischemic stroke. View Large Odds ratios for VT with different fasting homocysteine concentrations are presented in Table 3, for comparisons between patients and RDD controls, and Table 4 for comparisons between patients and partner controls. Overall, the age and sex-adjusted odds ratio for fasting homocysteine concentration in the comparison of patients with RDD controls was 1.01 (95% confidence interval (CI): 1.00, 1.02) when using plasma homocysteine concentrations as a continuous (per 0.13-mg/L increase) variable and 1.04 (95% CI: 0.98, 1.10) in terms of 0.7-mg/L increase, and 1.30 (95% CI: 1.02, 1.67) when the concentrations of ≥2.3 mg/L were compared with concentrations <1.6 mg/L. In the analysis of concentrations of ≥2.3 mg/L relative to concentrations of <1.6 mg/L, the odds ratio was 1.30 (95% CI; 1.02, 1.67) adjusted for age and sex, 1.27 (95% CI: 0.98,1.64) additionally adjusting for body mass index and smoking, 1.28 (95% CI: 0.99,1.65) after adding statin use, 1.19 (95% CI: 0.91,1.55) after adding cardiovascular disease, and 1.02 (95% CI: 0.77,1.34) after adding sports activities. Results from the 1:1-matched analysis between patients and partner controls resembled those from patients versus RDD controls (Table 4). In the analysis stratified by sex, on patients and RDD controls, crude and fully adjusted odds ratios remained close to unity (Table 5). Table 3. Risk of Venous Thrombosis According to Categories of Homocysteine Concentrations in All Patients and Random-Digit Dialing Controls, Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis, the Netherlands, 1999–2004 Homocysteine Concentration, mg/L  All Patients  RDD Controls  Model 1a  Model 2b  Model 3c  Model 4d  Model 5e  No.  %  No.  %  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  <1.60  888  53  518  55  1.00  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.60–1.79  250  15  154  16  0.95  0.76, 1.20  0.92  0.72, 1.16  0.91  0.72, 1.16  0.9  0.70, 1.14  0.86  0.66, 1.11  1.80–2.09  187  11  96  10  1.15  0.88, 1.51  1.11  0.84, 1.47  1.86  0.82, 1.44  1.03  0.77, 1.37  1.08  0.79, 1.48  2.10–2.29  103  6  54  6  1.10  0.78, 1.57  1.01  0.71, 1.45  0.97  0.68, 1.39  0.97  0.67, 1.39  0.92  0.62, 1.34  ≥2.30  261  15  117  13  1.30  1.02, 1.67  1.27  0.98, 1.64  1.28  0.99, 1.65  1.19  0.91, 1.55  1.02  0.77, 1.34  Continuous          1.01  1.00, 1.02  1.00  0.99, 1.02  1.01  0.99, 1.02  1.00  0.99, 1.01  1.00  0.99, 1.01  Per 0.7-mg/L increase          1.04  0.98, 1.10  1.03  0.97, 1.09  1.03  0.97, 1.09  1.02  0.96, 1.07  0.99  0.93, 1.05  Homocysteine Concentration, mg/L  All Patients  RDD Controls  Model 1a  Model 2b  Model 3c  Model 4d  Model 5e  No.  %  No.  %  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  <1.60  888  53  518  55  1.00  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.60–1.79  250  15  154  16  0.95  0.76, 1.20  0.92  0.72, 1.16  0.91  0.72, 1.16  0.9  0.70, 1.14  0.86  0.66, 1.11  1.80–2.09  187  11  96  10  1.15  0.88, 1.51  1.11  0.84, 1.47  1.86  0.82, 1.44  1.03  0.77, 1.37  1.08  0.79, 1.48  2.10–2.29  103  6  54  6  1.10  0.78, 1.57  1.01  0.71, 1.45  0.97  0.68, 1.39  0.97  0.67, 1.39  0.92  0.62, 1.34  ≥2.30  261  15  117  13  1.30  1.02, 1.67  1.27  0.98, 1.64  1.28  0.99, 1.65  1.19  0.91, 1.55  1.02  0.77, 1.34  Continuous          1.01  1.00, 1.02  1.00  0.99, 1.02  1.01  0.99, 1.02  1.00  0.99, 1.01  1.00  0.99, 1.01  Per 0.7-mg/L increase          1.04  0.98, 1.10  1.03  0.97, 1.09  1.03  0.97, 1.09  1.02  0.96, 1.07  0.99  0.93, 1.05  Abbreviations: BMI, body mass index; CI, confidence interval; OR, odds ratio. a Model 1 adjusted for age and sex. b Model 2 adjusted for age, sex, BMI, and smoking. c Model 3 adjusted for age, sex, BMI, smoking, and statin use. d Model 4 adjusted for age, sex, BMI, smoking, statin use, and cardiovascular disease. e Model 5 adjusted for age, sex, BMI, smoking, statin use, cardiovascular disease, and sports activity. Table 3. Risk of Venous Thrombosis According to Categories of Homocysteine Concentrations in All Patients and Random-Digit Dialing Controls, Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis, the Netherlands, 1999–2004 Homocysteine Concentration, mg/L  All Patients  RDD Controls  Model 1a  Model 2b  Model 3c  Model 4d  Model 5e  No.  %  No.  %  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  <1.60  888  53  518  55  1.00  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.60–1.79  250  15  154  16  0.95  0.76, 1.20  0.92  0.72, 1.16  0.91  0.72, 1.16  0.9  0.70, 1.14  0.86  0.66, 1.11  1.80–2.09  187  11  96  10  1.15  0.88, 1.51  1.11  0.84, 1.47  1.86  0.82, 1.44  1.03  0.77, 1.37  1.08  0.79, 1.48  2.10–2.29  103  6  54  6  1.10  0.78, 1.57  1.01  0.71, 1.45  0.97  0.68, 1.39  0.97  0.67, 1.39  0.92  0.62, 1.34  ≥2.30  261  15  117  13  1.30  1.02, 1.67  1.27  0.98, 1.64  1.28  0.99, 1.65  1.19  0.91, 1.55  1.02  0.77, 1.34  Continuous          1.01  1.00, 1.02  1.00  0.99, 1.02  1.01  0.99, 1.02  1.00  0.99, 1.01  1.00  0.99, 1.01  Per 0.7-mg/L increase          1.04  0.98, 1.10  1.03  0.97, 1.09  1.03  0.97, 1.09  1.02  0.96, 1.07  0.99  0.93, 1.05  Homocysteine Concentration, mg/L  All Patients  RDD Controls  Model 1a  Model 2b  Model 3c  Model 4d  Model 5e  No.  %  No.  %  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  <1.60  888  53  518  55  1.00  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.60–1.79  250  15  154  16  0.95  0.76, 1.20  0.92  0.72, 1.16  0.91  0.72, 1.16  0.9  0.70, 1.14  0.86  0.66, 1.11  1.80–2.09  187  11  96  10  1.15  0.88, 1.51  1.11  0.84, 1.47  1.86  0.82, 1.44  1.03  0.77, 1.37  1.08  0.79, 1.48  2.10–2.29  103  6  54  6  1.10  0.78, 1.57  1.01  0.71, 1.45  0.97  0.68, 1.39  0.97  0.67, 1.39  0.92  0.62, 1.34  ≥2.30  261  15  117  13  1.30  1.02, 1.67  1.27  0.98, 1.64  1.28  0.99, 1.65  1.19  0.91, 1.55  1.02  0.77, 1.34  Continuous          1.01  1.00, 1.02  1.00  0.99, 1.02  1.01  0.99, 1.02  1.00  0.99, 1.01  1.00  0.99, 1.01  Per 0.7-mg/L increase          1.04  0.98, 1.10  1.03  0.97, 1.09  1.03  0.97, 1.09  1.02  0.96, 1.07  0.99  0.93, 1.05  Abbreviations: BMI, body mass index; CI, confidence interval; OR, odds ratio. a Model 1 adjusted for age and sex. b Model 2 adjusted for age, sex, BMI, and smoking. c Model 3 adjusted for age, sex, BMI, smoking, and statin use. d Model 4 adjusted for age, sex, BMI, smoking, statin use, and cardiovascular disease. e Model 5 adjusted for age, sex, BMI, smoking, statin use, cardiovascular disease, and sports activity. Table 4. Risk of Venous Thrombosis According to Categories of Homocysteine Concentrations in Patients With Partners and Partner Controls, Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis, the Netherlands, 1999–2004 Homocysteine Concentration, mg/L  Patients With Partners  Partner Controls  Model 1a  Model 2b  Model 3c  Model 4d  Model 5e  No.  %  No.  %  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  <1.60  443  55  449  57  1.0  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.60–1.79  106  13  117  15  0.96  0.71, 1.29  0.95  0.70, 1.3  0.95  0.69, 1.30  1.05  0.76, 1.46  1.01  0.70, 1.44  1.80–2.09  80  10  76  10  1.10  0.77, 1.57  1.10  0.76, 1.61  1.09  0.75, 1.59  1.13  0.76, 1.70  1.33  0.86, 2.06  2.10–2.29  52  7  52  6  1.10  0.72, 1.68  1.13  0.73, 1.75  1.09  0.70, 1.70  1.24  0.77, 1.97  1.16  0.70, 1.94  ≥2.30  116  15  93  12  1.36  0.97, 1.92  1.40  0.97, 2.01  1.38  0.96, 1.99  1.38  0.94, 2.02  1.44  0.95, 2.17  Continuous          1.01  0.99, 1.02  1.01  0.99, 1.03  1.01  0.99, 1.03  1.01  0.99, 1.03  1.01  0.99, 1.03  Per 0.7-mg/L          1.05  0.97, 1.13  1.05  0.97, 1.14  1.05  0.97, 1.14  1.05  0.97, 1.14  1.05  0.96, 1.15  Homocysteine Concentration, mg/L  Patients With Partners  Partner Controls  Model 1a  Model 2b  Model 3c  Model 4d  Model 5e  No.  %  No.  %  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  <1.60  443  55  449  57  1.0  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.60–1.79  106  13  117  15  0.96  0.71, 1.29  0.95  0.70, 1.3  0.95  0.69, 1.30  1.05  0.76, 1.46  1.01  0.70, 1.44  1.80–2.09  80  10  76  10  1.10  0.77, 1.57  1.10  0.76, 1.61  1.09  0.75, 1.59  1.13  0.76, 1.70  1.33  0.86, 2.06  2.10–2.29  52  7  52  6  1.10  0.72, 1.68  1.13  0.73, 1.75  1.09  0.70, 1.70  1.24  0.77, 1.97  1.16  0.70, 1.94  ≥2.30  116  15  93  12  1.36  0.97, 1.92  1.40  0.97, 2.01  1.38  0.96, 1.99  1.38  0.94, 2.02  1.44  0.95, 2.17  Continuous          1.01  0.99, 1.02  1.01  0.99, 1.03  1.01  0.99, 1.03  1.01  0.99, 1.03  1.01  0.99, 1.03  Per 0.7-mg/L          1.05  0.97, 1.13  1.05  0.97, 1.14  1.05  0.97, 1.14  1.05  0.97, 1.14  1.05  0.96, 1.15  Abbreviations: BMI, body mass index; CI, confidence interval; OR, odds ratio. a Model 1 adjusted for age and sex, and partnership. b Model 2 adjusted for age, sex, BMI, smoking, and partnership. c Model 3 adjusted for age, sex, BMI, smoking, statin use, and partnership. d Model 4 adjusted for age, sex, BMI, smoking, statin use, cardiovascular disease, and partnership. e Model 5 adjusted for age, sex, BMI, smoking, statin use, cardiovascular disease, sports activity, and partnership. Table 4. Risk of Venous Thrombosis According to Categories of Homocysteine Concentrations in Patients With Partners and Partner Controls, Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis, the Netherlands, 1999–2004 Homocysteine Concentration, mg/L  Patients With Partners  Partner Controls  Model 1a  Model 2b  Model 3c  Model 4d  Model 5e  No.  %  No.  %  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  <1.60  443  55  449  57  1.0  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.60–1.79  106  13  117  15  0.96  0.71, 1.29  0.95  0.70, 1.3  0.95  0.69, 1.30  1.05  0.76, 1.46  1.01  0.70, 1.44  1.80–2.09  80  10  76  10  1.10  0.77, 1.57  1.10  0.76, 1.61  1.09  0.75, 1.59  1.13  0.76, 1.70  1.33  0.86, 2.06  2.10–2.29  52  7  52  6  1.10  0.72, 1.68  1.13  0.73, 1.75  1.09  0.70, 1.70  1.24  0.77, 1.97  1.16  0.70, 1.94  ≥2.30  116  15  93  12  1.36  0.97, 1.92  1.40  0.97, 2.01  1.38  0.96, 1.99  1.38  0.94, 2.02  1.44  0.95, 2.17  Continuous          1.01  0.99, 1.02  1.01  0.99, 1.03  1.01  0.99, 1.03  1.01  0.99, 1.03  1.01  0.99, 1.03  Per 0.7-mg/L          1.05  0.97, 1.13  1.05  0.97, 1.14  1.05  0.97, 1.14  1.05  0.97, 1.14  1.05  0.96, 1.15  Homocysteine Concentration, mg/L  Patients With Partners  Partner Controls  Model 1a  Model 2b  Model 3c  Model 4d  Model 5e  No.  %  No.  %  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  OR  95% CI  <1.60  443  55  449  57  1.0  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.00  Referent  1.60–1.79  106  13  117  15  0.96  0.71, 1.29  0.95  0.70, 1.3  0.95  0.69, 1.30  1.05  0.76, 1.46  1.01  0.70, 1.44  1.80–2.09  80  10  76  10  1.10  0.77, 1.57  1.10  0.76, 1.61  1.09  0.75, 1.59  1.13  0.76, 1.70  1.33  0.86, 2.06  2.10–2.29  52  7  52  6  1.10  0.72, 1.68  1.13  0.73, 1.75  1.09  0.70, 1.70  1.24  0.77, 1.97  1.16  0.70, 1.94  ≥2.30  116  15  93  12  1.36  0.97, 1.92  1.40  0.97, 2.01  1.38  0.96, 1.99  1.38  0.94, 2.02  1.44  0.95, 2.17  Continuous          1.01  0.99, 1.02  1.01  0.99, 1.03  1.01  0.99, 1.03  1.01  0.99, 1.03  1.01  0.99, 1.03  Per 0.7-mg/L          1.05  0.97, 1.13  1.05  0.97, 1.14  1.05  0.97, 1.14  1.05  0.97, 1.14  1.05  0.96, 1.15  Abbreviations: BMI, body mass index; CI, confidence interval; OR, odds ratio. a Model 1 adjusted for age and sex, and partnership. b Model 2 adjusted for age, sex, BMI, smoking, and partnership. c Model 3 adjusted for age, sex, BMI, smoking, statin use, and partnership. d Model 4 adjusted for age, sex, BMI, smoking, statin use, cardiovascular disease, and partnership. e Model 5 adjusted for age, sex, BMI, smoking, statin use, cardiovascular disease, sports activity, and partnership. Table 5. Risk of Venous Thrombosis in Men and Women According to Categories of Homocysteine Concentration, Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis, the Netherlands, 1999–2004 Homocysteine Concentration, mg/L  All Patients  RDD Controls  Adjusted for Age and Sex  Adjusted for Age, Sex, and Other Factorsa  No.  %  No.  %  OR  95% CI  OR  95% CI  Men   <1.60  370  44  241  49  1.00  Referent  1.00  Referent   1.60–1.79  143  17  89  18  1.01  0.74, 1.39  1.01  0.71, 1.44   1.80–2.09  123  14  57  12  1.36  0.96, 1.95  1.34  0.89, 2.03   2.10–2.29  58  7  28  6  1.25  0.77, 2.03  1.13  0.66, 1.93   ≥2.30  156  18  76  15  1.26  0.92, 1.74  1.08  0.75, 1.57  Women   <1.60  518  62  227  62  1.00  Referent  1.00  Referent   1.60–1.79  107  13  65  14  0.89  0.63, 1.25  0.75  0.48, 1.15   1.80–2.09  64  8  37  9  0.88  0.58, 1.35  0.76  0.45, 1.29   2.10–2.29  45  5  26  6  0.95  0.57, 1.58  0.77  0.42, 1.43   ≥2.30  105  12  41  9  1.41  0.95, 2.08  0.96  0.59, 1.54  Homocysteine Concentration, mg/L  All Patients  RDD Controls  Adjusted for Age and Sex  Adjusted for Age, Sex, and Other Factorsa  No.  %  No.  %  OR  95% CI  OR  95% CI  Men   <1.60  370  44  241  49  1.00  Referent  1.00  Referent   1.60–1.79  143  17  89  18  1.01  0.74, 1.39  1.01  0.71, 1.44   1.80–2.09  123  14  57  12  1.36  0.96, 1.95  1.34  0.89, 2.03   2.10–2.29  58  7  28  6  1.25  0.77, 2.03  1.13  0.66, 1.93   ≥2.30  156  18  76  15  1.26  0.92, 1.74  1.08  0.75, 1.57  Women   <1.60  518  62  227  62  1.00  Referent  1.00  Referent   1.60–1.79  107  13  65  14  0.89  0.63, 1.25  0.75  0.48, 1.15   1.80–2.09  64  8  37  9  0.88  0.58, 1.35  0.76  0.45, 1.29   2.10–2.29  45  5  26  6  0.95  0.57, 1.58  0.77  0.42, 1.43   ≥2.30  105  12  41  9  1.41  0.95, 2.08  0.96  0.59, 1.54  Abbreviations: CI, confidence interval; OR, odds ratio; RDD, random-digit dialing. a Additionally adjusted for body mass index, smoking, statin use, cardiovascular disease, sports activity, and (in women) hormone use. Table 5. Risk of Venous Thrombosis in Men and Women According to Categories of Homocysteine Concentration, Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis, the Netherlands, 1999–2004 Homocysteine Concentration, mg/L  All Patients  RDD Controls  Adjusted for Age and Sex  Adjusted for Age, Sex, and Other Factorsa  No.  %  No.  %  OR  95% CI  OR  95% CI  Men   <1.60  370  44  241  49  1.00  Referent  1.00  Referent   1.60–1.79  143  17  89  18  1.01  0.74, 1.39  1.01  0.71, 1.44   1.80–2.09  123  14  57  12  1.36  0.96, 1.95  1.34  0.89, 2.03   2.10–2.29  58  7  28  6  1.25  0.77, 2.03  1.13  0.66, 1.93   ≥2.30  156  18  76  15  1.26  0.92, 1.74  1.08  0.75, 1.57  Women   <1.60  518  62  227  62  1.00  Referent  1.00  Referent   1.60–1.79  107  13  65  14  0.89  0.63, 1.25  0.75  0.48, 1.15   1.80–2.09  64  8  37  9  0.88  0.58, 1.35  0.76  0.45, 1.29   2.10–2.29  45  5  26  6  0.95  0.57, 1.58  0.77  0.42, 1.43   ≥2.30  105  12  41  9  1.41  0.95, 2.08  0.96  0.59, 1.54  Homocysteine Concentration, mg/L  All Patients  RDD Controls  Adjusted for Age and Sex  Adjusted for Age, Sex, and Other Factorsa  No.  %  No.  %  OR  95% CI  OR  95% CI  Men   <1.60  370  44  241  49  1.00  Referent  1.00  Referent   1.60–1.79  143  17  89  18  1.01  0.74, 1.39  1.01  0.71, 1.44   1.80–2.09  123  14  57  12  1.36  0.96, 1.95  1.34  0.89, 2.03   2.10–2.29  58  7  28  6  1.25  0.77, 2.03  1.13  0.66, 1.93   ≥2.30  156  18  76  15  1.26  0.92, 1.74  1.08  0.75, 1.57  Women   <1.60  518  62  227  62  1.00  Referent  1.00  Referent   1.60–1.79  107  13  65  14  0.89  0.63, 1.25  0.75  0.48, 1.15   1.80–2.09  64  8  37  9  0.88  0.58, 1.35  0.76  0.45, 1.29   2.10–2.29  45  5  26  6  0.95  0.57, 1.58  0.77  0.42, 1.43   ≥2.30  105  12  41  9  1.41  0.95, 2.08  0.96  0.59, 1.54  Abbreviations: CI, confidence interval; OR, odds ratio; RDD, random-digit dialing. a Additionally adjusted for body mass index, smoking, statin use, cardiovascular disease, sports activity, and (in women) hormone use. The risk estimates for extremely high concentrations of homocysteine (in mg/L: 2.30–3.19, 3.20–6.40, and >6.40) are presented in Web Table 5. Crude and fully adjusted odds ratios for VT yielded null associations in all homocysteine categories, both for analyses with RDD controls and with partner controls. The highest (fully adjusted) odds ratio we found was in the matched analysis between patients and partner controls in the category of homocysteine concentrations between 3.2 and 6.4 mg/L, and it was 1.83 (95% CI: 0.95, 3.51). We investigated the association of homocysteine concentrations for subgroups of patients, restricting the analysis to provoked or unprovoked VT and DVT or pulmonary embolism (Web Table 6). A weak association was found between homocysteine concentrations higher than 2.3 mg/L and provoked VT as well as with DVT (age and sex–adjusted odds ratios were 1.37 (95% CI: 1.03, 1.83) and 1.34 (95% CI: 1.04, 1.74), respectively) when comparing patients with RDD controls. However, these findings were attenuated after adjustment for multiple confounding factors (for the provoked VT group, odds ratio = 1.08, 95% CI: 0.77, 1.50; for the DVT group, odds ratio = 1.15, 95% CI: 0.85, 1.56). Other odds ratios from this analysis were close to unity. DISCUSSION We analyzed data from the MEGA case-control study to investigate the association among different homocysteine concentrations and VT risk. We used 2 different control groups to study this association, population-derived controls identified using RDD and patients’ partner controls. Results from these 2 separate analyses demonstrated that hyperhomocysteinemia is not associated with increased risk of VT. The results were consistent when we used plasma homocysteine concentrations as a continuous variable and in terms of 0.7-mg/L increases. Any potential dose-response or threshold outcome was excluded. Results were consistent in men, women, patients with unprovoked or provoked VT, and in patients who had DVT or pulmonary embolism as the initial event. Our findings contradicted the results from 3 previous meta-analyses that reported hyperhomocysteinemia to be a risk factor for VT (4–6). The most recent meta-analysis included 3,289 patients and 3,780 controls from retrospective follow-up studies and 476 patients and 1,517 controls from prospective follow-up studies. The risks of VT per 0.7-mg/L (5 μmol/L) increase in plasma homocysteine were 1.60 (95% CI: 1.10, 2.34) and 1.27 (95% CI: 1.01, 1.59), respectively (4). However, the major problem of these meta-analyses is the possibility of confounding effects and publication bias. Our study included 1,689 patients and 1,726 controls. Moreover, we were able to adjust for many confounding factors not included in other studies due to small sample sizes. Additionally, we were able to exclude an association among homocysteine concentrations and DVT, pulmonary embolism, provoked VT, and unprovoked VT in the subgroup analyses. Our findings are in line with previous publication from the MEGA study, which reported no association between MTHFR genotype and VT (22). Because the MTHFR 677C→T variant has a phenotype in which homocysteine concentration is genetically increased, this finding provides further evidence that the association observed in previous studies cannot be causal. Two studies found sex differences in the association. The second Norwegian Health Study of Nord-Trøndelag (HUNT2) found that elevated homocysteine concentrations in men increased the frequency of a subsequent first VT 2-fold, whereas in women there was no relationship (8). In contrast, the Longitudinal Investigation of Thromboembolism Etiology (LITE) study, found that the association was higher in women (23). In MEGA, we did not find an association with VT in any of the postulated homocysteine concentrations when we analyzed men and women separately. Our null findings and understanding of the role of homocysteine in venous thrombotic disease are in line with the evolution of evidence for homocysteine as a potential target to modify arterial vascular disease risk. There too a promising hypothesis was first sparked by small retrospective studies in the 1990s (that is, a 2-fold increased risk of cardiovascular disease when homocysteine concentration was elevated) but returned to the sobering conclusion in 2012 (no increased risk) when all the evidence from published and unpublished studies was meta-analyzed with individual patient data and performing multiple adjustment for confounding factors (24). Although the MEGA study included a large number of individuals, we could not estimate in detail the risk of VT when the concentration of homocysteine was higher than 6.4 mg/L (i.e., concentrations that are associated with homocystinuria) given that n values were small (n = 16 patients, n = 7 RDD controls, and n = 6 partners). However, in one collaborative study, in which 5 centers from Ireland, Australia, the Netherlands, and the United States participated, information from patients with homocystinuria due to cystathionine β-synthase deficiency was registered, and a 90% reduction in vascular events was found after B-vitamin supplementation during an average treatment time of 17.9 years per patient (25). Patients with this diagnosis are expected to have plasma homocysteine concentrations above 6.4 mg/L, usually in the range of 13–65 mg/L (26). In terms of clinical implications, the present study, together with a previous study from MEGA on B-vitamin supplementation and VT risk (12), provides no evidence that B-vitamin supplementation will decrease VT risk in patients with plasma homocysteine concentrations below 6.4 mg/L without a diagnosis of homozygous cystathionine β-synthase deficiency. Certain limitations in this study should be noted. First, we excluded participants who were using B-vitamin supplementation, because vitamin-B intake lowers homocysteine concentrations (10, 11). Therefore, our results apply only to individuals who are not using B-vitamin therapy. Second, blood samples were collected in the outpatient setting, which could lead to an underrepresentation of chronically ill and bedridden patients. Third, our study was performed in individuals aged 18–70 years in a population that is mainly white in origin. Our results may be generalizable only to those who are well enough to visit an outpatient clinic and might not apply to the elderly and to other ethnicities. Fourth, there was no repeated or confirmatory capture of self-reported variables or measurement of fasting blood values, which might have led to misclassification in the study. Fifth, although our study is, to our knowledge, the largest case-control study on this issue to date, numbers in some subgroups were small, leading to large confidence intervals. Sixth, the MEGA study is a case-control study in which homocysteine concentrations were measured after the event. Whether these homocysteine concentrations are representative of levels before the event cannot be said with certainty. A previous meta-analysis found that of the 32 studies published at the time, 29 studies had homocysteine measurements from after the venous thrombotic event and only 3 studies had homocysteine measurements from prior to the event (4). The same meta-analysis showed that the association was stronger in the pooled retrospective studies than in the pooled prospective studies, and although this can be explained by several factors (such as regression dilution bias (27), an underestimation of the strength of the association as a certain exposure value at the beginning of follow-up might have changed at the time of event onset), reverse causation is likely to have played a role here. The fact that we found no association, in contrast to the previous retrospective studies, suggests that reverse causation was not present in our study. This may have been a result of 2 measures we took in the design of the study: 1) Measure plasma homocysteine concentration at least 3 months after VT occurred (we assume that an acute effect of VT leading to an increase in homocysteine concentration must have worn off); 2) exclude all pregnant women (who are folic acid users) and all individuals who reported B-vitamin supplementation at the time of blood draw (because B vitamins can lower homocysteine levels) from the study. Seventh, choosing controls in case-control studies can be difficult and can influence results. In the MEGA study, the partner controls may have been too closely matched to patients and could therefore yield null results, although conditional logistic regression should take this into account. In addition, RDD controls may be too healthy and therefore yield spurious results, given that health is related to lower homocysteine concentrations. A strength of the MEGA study is that both types of controls were included and that results from the study were independent of the type of control in our analyses. Eighth, because this is a matched case-control study, results could be contingent based on the single match that was made. The main message from this study is that when performing an observational study on the association of a certain biomarker with a specific disease, it is important to consider the drug therapy that can modify the biomarker concentration and other confounding factors that can bias the results. Building on this logic, the present observational study yielded the same results as randomized clinical trials that balance measured and unmeasured confounders. This study confirms what randomized clinical trials have shown: no association between homocysteine-lowering therapy and risk of VT (10, 11). In conclusion, in this study there was no evidence of an association among different homocysteine concentrations and increased risks for VT, whether for DVT, pulmonary embolism, provoked or unprovoked VT, or in men or women. ACKNOWLEDGMENTS Author affiliations: Department of Clinical Epidemiology, Leiden University Medical Center, Leiden, the Netherlands (Monica Ospina-Romero, Suzanne C. Cannegieter, Martin den Heijer, Carine J. M. Doggen, Frits. R. Rosendaal, Willem M. Lijfering); Department of Epidemiology and Biostatistics, University of California San Francisco, San Francisco, California (Monica Ospina-Romero); Department of Thrombosis and Hemostasis, Leiden University Medical Center, Leiden, the Netherlands (Suzanne C. Cannegieter); Department of Internal Medicine, VU Medical Center, Amsterdam, the Netherlands (Martin den Heijer); Department of Health Technology and Services Research, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, the Netherlands (Carine J. M. Doggen); and Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands (Frits. R. Rosendaal, Willem M. Lijfering). This research was supported by the Netherlands Heart Foundation (grant NHS 98.113), the Dutch Cancer Foundation (grant RUL 99/1992), and the Netherlands Organization for Scientific Research (grant 912-03-033 2003). The Netherlands Heart Foundation, the Dutch Cancer Foundation, and the Netherlands Organization for Scientific Research had no role in the design and conduct of the study; the collection, management, analysis, and interpretation of the data; or the preparation, review, or approval of the manuscript. Conflict of interest: none declared. Abbreviations CI confidence interval DVT deep vein thrombosis MEGA Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis RDD random digit-dialing VT venous thrombosis REFERENCES 1 ISTH Steering Committee for World Thrombosis Day. Thrombosis: a major contributor to the global disease burden. J Thromb Haemost . 2014; 12( 10): 1580– 1590. CrossRef Search ADS PubMed  2 Ray JG. Hyperhomocysteinemia: no longer a consideration in the management of venous thromboembolism. Curr Opin Pulm Med . 2008; 14( 5): 369– 373 Google Scholar CrossRef Search ADS PubMed  3 McCully KS. Vascular pathology of homocysteinemia: implications for the pathogenesis of arteriosclerosis. Am J Pathol . 1969; 56( 1): 111– 128 Google Scholar PubMed  4 den Heijer M, Lewington S, Clarke R. Homocysteine, MTHFR and risk of venous thrombosis: a meta-analysis of published epidemiological studies. J Thromb Haemost . 2005; 3( 2): 292– 299. 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American Journal of EpidemiologyOxford University Press

Published: Jan 12, 2018

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