Genetic variation in heme oxygenase 1 (HMOX1) and the risk of recurrent venous thromboembolism
Article Outline
Background/Objective
Products of heme oxygenase 1 (HO1) possess antithrombotic properties, and impairment of HO1 activity may contribute to thrombus formation. Transcriptional activity of long GT-repeat alleles in HO1 gene (HMOX1) is lower as compared with short alleles. We hypothesize that these long alleles are associated with decreased HO1 anticoagulant activity and, thus, an increased risk of thrombosis..
Design/Methods
In a prospective cohort study, we followed 860 patients with a first VTE, and investigated the impact of a promoter GT-dinucleotid length polymorphism in HOMX1 on the risk of recurrent venous thromboembolism (VTE).
Results
Allele groups short (S), medium (M) and long (L) of the promoter GT-dinucleotide length polymorphism were distinguished. L-alleles, but not M- or S-alleles, were found to be more frequent among patients with recurrence. Heterozygous carriers of L-alleles had a two-fold higher relative risk of recurrence [(RR 2.2 (95% CI: 1.4-3.4)] as compared to wild type, which was independent of other thrombotic risk factors. At five years, the cumulative probability of recurrence was 18% (95% CI: 15%-22%) in patients without an L-allele compared to 32% (95% CI: 19%-46%) in patients heterozygous for the L-allele (P = .001).
Conclusion
Patients with first VTE and long GT-repeat alleles in HMOX1 have an increased risk of recurrence. Genetically determined alterations in HO1 function may represent a new pathomechanism in VTE.
Venous thromboembolism (VTE) is a chronic and multicausal disease.1 The annual incidence of recurrence ranges between 5% to 10%, and approximately 5% of patients with recurrence die from pulmonary embolism. The risk of VTE depends upon the severity and number of risk factors in an individual patient at a given point in time. Over the years, numerous circumstantial and congenital risk factors of first VTE have been identified. The relevance of these factors with regard to stratifying patients according to their risk of recurrence is modest. Importantly, many patients with recurrent VTE carry neither a clinical nor thrombophilic risk factor. These findings suggest the existence of so far unknown risk factors of recurrent VTE.
Experimental data indicate that products of heme oxygenase 1 (HO1) activity possess antithrombotic properties, and impairment of HO1 activity may contribute to thrombus formation. HO1, the rate-limiting enzyme of heme degradation, cleaves the heme ring at the alpha methene bridge to form carbon monoxide (CO), ionic iron (Fe2+), and biliverdin, which is immediately converted to bilirubin by biliverdin reductase. By conversion of pro-oxidant heme into antioxidant bilirubin, HO1 acts as a major antioxidant.2, 3 Bilirubin, as well as systemic induction of HO1, delays microvascular thrombus formation in vivo, probably by a reduction of endothelial P-selectin.4 CO exerts anticoagulant effects by affecting fibrinolysis,5 platelet aggregation,6, 7, 8, 9 and by maintaining the integrity of the vessel wall.10, 11
The allele frequency of a polymorphic GT-repeat (g.15167397GT (23_37); dbSNP rs3074372) in the promoter of the HO1 gene (HMOX1) shows a trimodal distribution with peaks at 23 repeats, 30 repeats, and above 33 repeats. Accordingly, alleles have been referred to as short (S), medium (M), and long (L). Level of lipid peroxidation in vivo and clinical conditions like restenosis after percutaneous transluminal angioplasty as well as susceptibility to coronary artery disease in diabetes are related to HMOX1 genotype.12, 13, 14, 15 Importantly, transcriptional activity as determined by luciferase reporter assays of long GT-repeat alleles is lower compared with short alleles.12, 16
We hypothesize that prevalence of long GT-repeat alleles in HMOX1 leads to impaired HO1 anticoagulant activity and is associated with increased risk of thrombosis. In patients with venous thromboembolic disease, the role of HMOX1 genotype has not been investigated so far. In a large cohort study, we prospectively followed patients with a first spontaneous VTE and evaluated the risk of recurrence in association with the polymorphic GT-repeat in the promoter of the HMOX1 gene.
Methods and material
Study population
Patients enrolled in the Austrian Study on Recurrent Venous Thromboembolism (AUREC), an ongoing, prospective, multicenter cohort study, were eligible for the present analysis. The characteristics of the study have been previously reported in detail.17 To fulfill the inclusion criteria, patients had to be older than 18 years and had to be treated with vitamin K-antagonists for at least 3 months after VTE. Diagnosis of deep vein thrombosis had to be objectified by venography or color duplex sonography (in case of proximal deep vein thrombosis), and pulmonary embolism had to be diagnosed by ventilation perfusion scan according to the criteria of the prospective investigation of pulmonary embolism diagnosis or by multi-slice computed tomography. Between July 1992 and July 2004, 3006 patients with VTE were screened. Two thousand and seventy-one (2071) patients were excluded because of previous VTE, VTE secondary to surgery, trauma, or pregnancy, deficiency of a natural coagulation inhibitor, the lupus anticoagulant, cancer, and long-term antithrombotic treatment for reasons other than VTE. In 75 patients (8%) of the remaining 935 patients, no valid results of HMOX1 genotyping could be obtained.
Patients entered the study at the time of discontinuation of vitamin K-antagonists, and were seen at 3-month intervals during the first year and every 6 months thereafter. All women were strongly discouraged from hormone replacement therapy or contraceptive pill use. Patients received written information on the symptoms of VTE and were instructed to report if symptoms occurred.
The study has been approved by the ethics committee of the Medical University of Vienna and the patients had to provide written informed consent prior to participation. Data analyses were performed according to guidelines for describing DNA polymorphism-disease associations.18
Study endpoint
The endpoint of the study was recurrent, symptomatic deep vein thrombosis confirmed by venography or color duplex sonography (in case of proximal deep vein thrombosis of the contra lateral leg), or symptomatic pulmonary embolism verified by perfusion lung scan and/or multi-slice computed tomography.
Deep vein thrombosis was considered to have recurred if the patient had a thrombus in the leg or arm not affected by the previous thromboembolic event; a thrombus in another deep vein in the leg or arm affected by the previous event; or a thrombus in the same venous system affected in the previous event, with proximal extension of the thrombus (if the upper limit of the original thrombus had been visible) or with a constant-filling defect surrounded by contrast medium (if the original thrombus had not been visible). The diagnosis was established by an adjudication committee consisting of independent clinicians and radiologists.
Blood sampling and laboratory analysis
Three weeks after discontinuation of vitamin K-antagonists, venous blood was obtained after overnight-fasting. The blood was placed in 1/10 volume of 0.11 mmol of trisodium citrate per liter, and centrifuged for 20 minutes at 2000 g. Plasma was stored at −80°C. Genomic DNA was isolated from leukocytes using standard methods. Thrombophilic risk factors, including factor V Leiden, prothrombin G20210A mutation, levels of factor VIII, antithrombin, protein C, protein S, and the presence of a lupus anticoagulant were determined, as previously reported.17 In case a natural inhibitor deficiency, a lupus anticoagulant, homozygous, or double heterozygous mutations were detected at this time point, anticoagulant treatment was resumed and the patients were excluded from the study.
Genotyping was performed by personnel blinded for clinical data. For quality control genotyping was repeated in random samples and matching results were obtained in all cases (n = 70). Nucleotide positions relate to HMOX1 reference sequences NT_011520 for genomic sequences. Determination of the GT-Repeat located in the 5′-flanking region of the HO1 gene (g.15167397GT (23_37); dbSNP rs3074372) was done by polymerase chain reaction (PCR), using a fluorescent-labeled sense primer and an unlabeled antisense primer (5′-FAM-AGAGCCTGCAGCTTCTCAGA-3′; 5′-ACAAAGTCTGGCCATAGGAC-3′). PCR products were generated in 25 μl volumes containing 1.7 U of AmpliTaq Gold (Roche, Branchburg, NJ), 0.863 mmol/L MgCl2, 200 μmol/L of each dNTP (Amersham Pharmacia Biotech, Uppsala, Sweden), 4 pmol forward primer, 4 pmol reverse primer, and approximately 4 ng DNA. Amplifications were performed in 96-well plates together with several water controls placed in between on an Eppendorf Mastercycler (Eppendorf AG, Hamburg, Germany). A 10-minute denaturation period at 95°C was followed by 38 cycles of 93°C for 1 minute and 43°C for 5 minutes. A final extension step of 180 minutes at 72° completed the reaction. The length of the PCR products was determined relative to an internal size-standard (GeneScan ROX 350 size standard, Applied Biosystems, Foster City, Calif), after 5 minutes denaturation at 95° with a tenfold volume of Hi-Di formamide (Applied Biosystems, Foster City, Calif), on an automated DNA capillary sequencer (ABI Prism 3100 Genetic Analyser; Applied Biosystems, Foster City, Calif). Fragment length determination and GT-repeat length attribution was done semi automatically using ABI Prism Software (Gene Scan Analysis Version 3.7 and Genotyper Software Version 3.7, both Applied Biosystems, Foster City, Calif).
Statistical analysis
For numerical operations SPSS software (SPSS Inc. Headquarters, Chicago, Ill) was used. Values are given as means ± SD. To test for homogeneity between strata, we applied the log-rank and the generalized Wilcoxon test. Categorical data were checked for homogeneity with the use of contingency-table analyses (by the χ2 test if not indicated otherwise, or Fisher exact test). Simple descriptive statistics were computed to provide a clear presentation of data. For multivariate analyses, the data were adjusted for potential confounding thrombotic variables, such as age, sex, factor V Leiden mutation, factor II G20210A mutation, factor VIII, and body mass index (weight in kilograms by the square of height in meters) Times to recurrence (uncensored observations) or follow-up times in patients without recurrence (censored observations) were analyzed according to survival methods. The probability of recurrence was estimated according to the method of Kaplan and Meier.19
Results
Patients
Eight hundred and sixty (860) patients with a first spontaneous VTE were included in the analysis. Of these patients, 229 left the study during the course of the study because of a diagnosis of cancer (16 patients), because of pregnancy (33 patients), or because of antithrombotic therapy for reasons other than VTE (149 patients); 18 (2.14%) patients were lost to follow-up; 13 patients died, but in none recurrent VTE was the cause of death. Patients were followed until the time of exclusion or death, when data were censored.
VTE recurred in 134 (16%) of the 860 patients. Deep vein thrombosis occurred in 84 patients and pulmonary embolism in 50 patients; one of them was fatal. In 13 patients, recurrence was secondary to surgery (n = 7) or trauma (n = 6). Compared with patients without recurrence, patients with recurrence were slightly older (47 ± 16 years vs 50 ± 14 years, P = .1) and had higher levels of factor VIII (163 ± 46 IU/dL vs 178 ± 54 IU/dL P = .017). Women were less frequent among recurrences (30% vs 59%, P < .001). The proportion of carriers of factor V Leiden (34% vs 28%) or factor II G20210A (10% vs 7%) did not significantly differ between patients with and without recurrence.
Length of GT-repeats in HMOX1 in patients with VTE
Clustering the HMOX1 GT-repeat alleles into three groups, designated short (S; up to 29 repeats), medium (M; 30 to 33 repeats), and long (L; from 34 repeats upward), resulted in six genotype groups: SS, SM, SL, MM, ML, and LL (Table I). Allele frequencies for S-alleles, M-alleles, and L alleles were 44.9, 53.6, and 4.5%, respectively. The overall distribution of genotypes was in agreement with the Hardy Weinberg law (χ2 4.73; P [2df] = > .05).
Table I. Distribution of 860 patients with and without recurrence according to GT-repeat genotype groups
| SS | SM | SL | MM | ML | LL | |
|---|---|---|---|---|---|---|
| No recurrence (n = 726), no (%) | 142 (20) | 310(43) | 21(3) | 221(30) | 29(4) | 3(0) |
| Recurrence (n = 134), no (%) | 23(17) | 51(38) | 9(7) | 39(29) | 12(9) | 0(0) |
| All (n = 860), no (%) | 165(19) | 361(42) | 30(4) | 260(30) | 41(5) | 3(0) |
L-alleles and the risk of recurrent VTE
The frequencies of GT-repeat alleles in patients with and without recurrent are shown in Fig 1. Long GT-repeats of more than 33 repeats were more frequent among patients with recurrence than among those without recurrence (21 of 268[8%] vs. 56 of 1452[4%], P = .004). Also the six genotypes were not evenly distributed in patients with and without recurrence (Fisher exact test; P = .049; Table I). As expected from allele frequencies, genotypes containing L alleles (genotypes SL and ML) were more frequent among patients with recurrence, while other genotypes, particularly SS, SM, and MM, showed a similar distribution in the two groups. When discriminating L-alleles from other HMOX1-alleles, the frequency of homozygous L/L genotypes, heterozygous L-allele genotypes, and genotypes containing no L-allele was 0, 21, and 113 among 134 patients with recurrence, compared with 3, 50, and 673 among 726 patients without recurrent VTE (Fisher exact test; P = .006). Since the number of homozygous carriers of the L-allele (n = 3) was too small to perform adequate statistical analysis, these patients were excluded from further analysis.
Fig 1.
GT-repeat alleles in patients with and without recurrent venous thromboembolism. Frequency of GT-repeat alleles in patients with recurrent (black columns) and without recurrent VTE (grey columns).
The characteristics of all patients with heterozygous L-allele genotypes and of patients who did not carry an L-allele are shown in Table II. Differences between the two groups with regard to clinical and laboratory risk factors of venous thrombosis were not seen. Carriers of an L-allele had an increased risk of recurrent VTE. At 5 years, the cumulative probability of recurrence was 18% (95% CI: 15%-22%) in patients without an L-allele compared with 32% (95% CI: 19%-46%) in patients with one L-allele (log-rank test P = .001; Breslow test P = .02) (Fig 2). In those patients the relative risk (RR) of recurrence was 2.2 ([95% CI: 1.4 - 3.4]; P = .001) compared with non-carriers of the L-allele. Adjustment for potential confounding variables including age, sex, prothrombin 20210A, factor V Leiden, high factor VIII, and body mass index only slightly affected the RR (2.0 [95% CI:1.3-3.3]; P = .003).
Table II. Patient characteristics: heterozygous carriers of an L-allele compared with non-carriers
| no L-allele N = 786 | heterozygous L-allele N = 71 | P | |
|---|---|---|---|
| Female, no (%) | 432(55) | 35(49) | .4 |
| Age (y) | 48(36-60) | 49(38-62) | .3 |
| Site of first VTE | .03 | ||
| Deep vein thrombosis, no (%) | 450(57) | 29(41) | |
| Pulmonary embolism, no (%) | 336(43) | 42(59) | |
| Observation time (mo) | 39(20-66) | 33(16-67) | .4 |
| Duration of therapy (mo) | 6.5(5.9-7.6) | 6.4(5.7-7.5) | .9 |
| Factor V Leiden; heterozygous | 220(28) | 27(38) | .1 |
| Factor II G20210A; heterozygous | 59(7,5) | 7(10) | .5 |
| Factor VIII level, IU/dL | 161(132-195) | 173(140-198) | .2 |
| Factor IX level, IU/dL | 117(101-135) | 121(105-136) | .2 |
| Recurrent VTE | 113(14) | 21(30) | .001 |
Fig 2.
Kaplan Meier estimates of the risk of recurrent venous thromboembolism for heterozygous carriers of an L-allele compared with non-carriers.
Discussion
In 860 patients with a first spontaneous VTE, we investigated the role of polymorphic HMOX1 genotypes with regard to risk of recurrent VTE. HMOX1 promoter GT-repeat alleles longer than 33 repeats were twice as frequent among patients with recurrent VTE as among those without recurrence. Important, relative risk of recurrence after a first thrombotic event was increased twofold among patients carrying one long GT-repeat allele compared with patients with short and medium length alleles. This increased risk of recurrence was independent of other confounding variables, including age, sex, and laboratory thrombophilic risk factors. Only three patients were homozygous carriers of the long GT-repeat allele, and none of these patients had recurrent VTE. Although it is conceivable that homozygous carriers of the L-allele are more prone to recurrent thrombosis than heterozygotes, we could not show this effect most likely due to the small number of patients.
Genotypes containing only short or medium length alleles were evenly distributed between patients with and without recurrence. This is seemingly in contrast to findings in patients after peripheral angioplasty in which the presence of medium length alleles was associated with in increased risk of early restenosis.15 This discrepancies, however, could be explained by patient selection or by different functional features of HMOX1 alleles involved in the pathomechanism of arterial and venous thrombotic disease.
Our study is the first to provide clinical evidence for a potential role of HO1 activity in the pathogenesis of recurrent VTE in a large number of patients. The HMOX1 L-allele was independently associated with an increased risk of recurrent VTE. There are some indications for a role of HMOX1 polymorphisms in venous disease. Elevated oxidative stress, measured in type 2 diabetic patients as level of thiobarbituric acid reactive substances, was observed in carriers of L-alleles.12 Increased oxidative stress is associated with platelet activation, which can contribute to thrombus formation.20 Importantly, luciferase reporter assays indicate that the transcriptional activity of L-alleles is lower compared with S-alleles.12, 16 Lower HO1 activity may result in impaired antithrombotic action of its metabolites4, 5, 6, 7, 8, 9, 10, 11 including CO and bilirubin. CO down-regulates plasminogen activator inhibitor-1 in mononuclear phagocytes5 and inhibits platelet aggregation.6, 7, 8 CO also prevents thrombus formation by maintaining the integrity of the vessel wall through direct blocking of vascular cell apoptosis as well as inhibiting release of pro-apoptotic inflammatory cytokines from the vessel wall.10, 11 Increased apoptosis associated with the L-allele was reported in an in vitro model of oxidant-induced apoptosis in lymphoblastoid cell lines.21 Experimental data indicate that bilirubin delays microvascular thrombus formation in vivo by reducing levels of endothelial P-selectin.4 P-selectin is important for fibrin formation and thrombus growth by supporting platelet rolling, recruitment of platelets onto vascular injury sites, and inducing generation of tissue-factor bearing microparticles.22 Interestingly, our cohort patients with recurrent VTE had significantly higher levels of P-selectin than those without recurrence. Patients with high P-selectin levels had an almost twofold higher risk of recurrence compared with those with lower levels.23
Due to the design of our study, we could not evaluate whether HOMX1 polymorphisms are also a risk factor for a first VTE. Thus, the role of this polymorphism in patients with a first venous thrombotic event needs to be affirmed in adequately designed studies. Furthermore, the effects of the GT-length polymorphism, especially L-alleles, on HO1 activity need to be further elucidated by functional assays.
Our study in a large number of patients with a first VTE provides novel aspects in the pathogenesis of recurrent venous thrombotic disease. L-alleles of the promoter GT-repeat length polymorphism in HMOX1 were more frequent among patients with recurrent VTE compared with those without recurrence. Long alleles were an independent risk factor of recurrent VTE conferring a twofold increased risk. Our study generated the hypothesis that genetically determined alterations in HO1 function may represent a new pathomechanism in venous thrombotic disease. Patients at high risk of recurrent VTE could be identified by screening for L-alleles of the promoter GT-repeat length polymorphism in HMOX1 and thromboprophylactic treatment could be provided to these patients to avoid recurrent disease. Our findings, however, need to be confirmed by other clinical studies and in different patient populations.
Author contributions
The authors thank Tuende Kliegel for his excellent technical work and Markus Exner for his critical reading of the manuscript.
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Competition of interest: none.
PII: S0741-5214(07)01549-2
doi:10.1016/j.jvs.2007.09.060
© 2008 The Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.
