ORIGINAL STUDY
Hereditary Thrombophilic Factors in Glaucoma Mehmet A. Sekeroglu, MD,* Murat Irkec, MD,w Mehmet C. Mocan, MD,w and Mehmet Orhan, MDw
Purpose: To evaluate the hereditary thrombophilic factors in patients with primary open-angle glaucoma, exfoliative glaucoma, and exfoliation syndrome and to compare their results with those of healthy control subjects. Materials and Methods: The study included 75 patients [25 patients with primary open-angle glaucoma (group I), 25 patients with exfoliative glaucoma (group II), and 25 patients with exfoliation syndrome (group III)] and 25 healthy control subjects (group IV). Well-known hereditary thrombophilic factors including methylenetetrahydrofolate reductase (MTHFR) gene C677T mutation, prothrombin G20210A mutation, factor V Leiden mutation, activated protein C resistance, protein S, protein C, and antithrombin III activities, and homocysteine levels were measured in venous blood samples of all subjects. Results: Fifty-one males and 49 females were included in the study. The mean age of the patients was 67.8 ± 8.7 years (range, 46 to 87 y). There was no statistically significant difference with regard to the mean age (P = 0.057) and distribution of sex (P = 0.391) between the study groups. The difference of homocysteine, folate, vitamin B12, antithrombin III activity, protein C activity, free protein S activity, and activated protein C resistance were not statistically significant; and the number of subjects with MTHFR C677T, prothrombin G20210A, and factor V Leiden mutations were similar between the study groups. Conclusion: Our results suggest that there is no significant difference between the prothrombotic inherited risk factors of glaucomatous and nonglaucomatous subjects. Key Words: exfoliation, glaucoma, homocysteine, thrombosis
(J Glaucoma 2016;25:203–207)
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laucoma, which is one of the leading causes of irreversible blindness in the world, is a progressive multifactorial optic neuropathy characterized by optic nerve head excavation and retinal ganglion cell loss with corresponding visual field damages.1 Although intraocular pressure (IOP) is assumed to be the most significant risk factor for the development of glaucoma,2 the exact mechanism(s) leading to glaucomatous damage have not been clarified yet. In addition to increased IOP, vascular, immunologic, and neurotoxic factors have also been proposed in the pathogenesis of glaucoma. The pathogenic Received for publication October 26, 2013; accepted August 22, 2014. From the *Ulucanlar Eye Training and Research Hospital; and wDepartment of Ophthalmology, Hacettepe University Faculty of Medicine, Ankara, Turkey. The study was conducted in Department of Ophthalmology, Hacettepe University Faculty of Medicine, Ankara, Turkey. Disclosure: The authors declare no conflict of interest. Reprints: Mehmet A. Sekeroglu, MD, Ulucanlar Eye Training and Research Hospital, 06230, Ankara, Turkey (e-mail: msekeroglu@ yahoo.com). Copyright r 2014 Wolters Kluwer Health, Inc. All rights reserved. DOI: 10.1097/IJG.0000000000000148
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Volume 25, Number 2, February 2016
mechanisms other than increased IOP, especially the vascular factors, gained greater importance in recent years.3–5 Impaired microcirculation of the optic nerve head because of the vascular risk factors may be due to anatomic or functional abnormalities of these vessels. Although the underlying reasons for impaired microcirculation of the optic nerve head in patients with glaucoma are not clear, one possible pathogenetic mechanism may involve abnormalities in prothrombotic inherited risk factors such as the presence of methylenetetrahydrofolate reductase (MTHFR) C677T mutation,6 prothrombin G20210A mutation, factor V Leiden mutation,7 activated protein C resistance (APCR), deficiencies in protein S, protein C, and antithrombin III activities, and elevated homocysteine levels,8 all of which may lead to a greater tendency for thrombosis and may produce a hypercoagulable state which in turn may result in impaired microcirculation of the optic nerve head. Mild hyperhomocysteinemia has been shown to be associated with primary open-angle glaucoma (POAG) and secondary open-angle glaucoma due to exfoliation syndrome (XFS).9,10 Furthermore, significant elevated homocysteine levels have been observed in the aqueous humor of XFG.11 Homocysteine induces apoptotic cell death in retinal ganglion cells by overstimulation of N-methyl-Daspartate receptors and caspase-3 activation.12,13 The purpose of the present study was to evaluate the most common hereditary thrombophilic factors that produce a hypercoagulable state in patients with POAG, exfoliative glaucoma (XFG), and XFS and to compare these parameters with those of healthy control subjects.
MATERIALS AND METHODS The study was designed as a prospective, case-control study undertaken at a single university-based hospital. Informed consent was obtained from all patients and the study was carried out with approval from the Institutional Review Board/Ethics Committee. A total of 100 consecutive subjects [25 POAG (group I), 25 XFG (group II), 25 XFS (group III), and 25 healthy control subjects (group IV)] were recruited between January 2007 and December 2008 for this study. Only patients who fulfilled the selection criteria and gave written informed consent in line with the Declaration of Helsinki were included in the study. All patients underwent complete ophthalmologic examination including best-corrected Snellen visual acuity testing, slit-lamp examination, Goldmann applanation tonometry, ultrasound corneal pachymetry, gonioscopic evaluation, dilated fundus examination using a 90 D lens, and visual field evaluation with the 30-2 SITA-Standard algorithm (Humphrey Visual Field Analyzer; Humphrey Instruments Inc., San Leandro, CA). A detailed medical history was also obtained. www.glaucomajournal.com |
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The diagnosis of POAG was established upon the presence of consistently elevated IOP > 21 mm Hg, normal anterior segment and gonioscopic findings, characteristic optic disc changes, and glaucomatous visual field defects. The diagnosis of XFS was made on slit-lamp examination following mydriasis, as well as gonioscopic evaluation, and included the presence of exfoliation material on the pupillary margin and the anterior lens capsule and trabecular hyperpigmentation. The diagnosis of XFG was made when anterior segment findings of XFS accompanied an IOP > 21 mm Hg without treatment, typical optic nerve head changes, and visual field defects. Twenty-five consecutive subjects aged above 45 years with no history of ocular disease except for refractive errors or presbyopia, an IOPr21 mm Hg, a normal optic disc appearance, and who had no visual field defects were included as controls. The criteria for exclusion included ocular diseases besides refraction disorders, POAG, XFG, XFS, and a history of past ocular surgery; systemic diseases such as diabetes mellitus, cardiovascular disease, dyslipidemia, renal failure, malignancy, autoimmune diseases, hematological diseases, chronic obstructive pulmonary disease, uncontrolled arterial hypertension, and transient ischemic attack or stroke; those who use some medications (such as fibrates, carbamazepine, phenytoin, methotrexate, and trimethoprim), vitamin supplements, or alcohol that may affect plasma homocysteine levels; and those with a history of central nervous system disease (multiple sclerosis, optic nerve glioma, optic neuritis, optic nerve sheath meningioma, and intracranial tumors) that might have interfered with visual field testing. After at least 8 hours of fasting, fresh peripheral whole blood samples of all subjects were collected from the antecubital vein at the same time interval of the day (9 to 11 AM). Fasting blood glucose, liver and renal function tests, serum triglycerides and cholesterol, vitamin B12, folate, hemoglobin, and hematocrit levels were determined in all subjects. Hematological tests were carried out using STA analyser (Diagnostica Stago Asnieres, France) and included activated partial thromboplastin time-(aPTT) (STAC.K.Prest), antithrombin III activity-AT III (chromogenic substrate assay; STA-AT III), protein C activity (synthetic chromogenic substrate method; STA-Protein C), and free protein S activity (immunoturbidimetric method; STALiatest Free Protein S) assays. Serum homocysteine levels were determined with a fluorescence polarization immunoassay (AXSYM-Homocysteine, AXSYM System; Abbott, Wiesbaden, Germany).
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For the analysis of MTHFR C677T, prothrombin G20210A, and factor V Leiden mutations, genomic DNA was extracted from 4 mL of EDTA anticoagulated blood by conventional phenol-chloroform method. Polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) was used for genotype analysis as mentioned in detail elsewhere.14–16 Statistical analyses were performed using SPSS version 15.0 (SPSS Inc., Chicago, IL) software. One-way analysis of variance (ANOVA) was used to compare quantitative data between multiple groups when a normal distribution was found and Kruskal-Wallis 1-way ANOVA on ranks was used if data were not normally distributed. Kruskal-Wallis Z-value test was used for all pairwise multiple comparisons. Student unpaired t test or the Mann-Whitney U test was used to compare quantitative data and w2 analysis was used for qualitative data. For all evaluations, P < 0.05 was considered significant. Post hoc calculation of statistical power was performed using NCSS-PASS software (NCSS, UT).
RESULTS A total of 100 subjects [25 patients with POAG (group I), 25 patients with XFG (group II), 25 patients with XFS (group III), and 25 healthy control subjects (group IV)] were included in the study. The mean age of the patients was 67.8 ± 8.7 years (range, 46 to 87 y). Of the 100 subjects, 51 were males and 49 were females. The mean age of group I was 64.4 ± 8.5 years (10 males, 15 females), group II was 68.9 ± 9.1 years (16 males, 9 females), group III was 70.8 ± 7.0 years (13 males, 12 females), and group IV was 67.3 ± 9.2 years (12 males, 13 females). There was no statistically significant difference with regard to the mean age (P = 0.057) and distribution of sex (P = 0.391) between the study groups. The complete blood counts including platelets were within normal limits in all subjects. None of the participants had elevated prothrombin levels and aPTT. The difference of homocysteine, folate, vitamin B12, aPTT, antithrombin III activity, protein C activity, free protein S activity, and APCR levels were not statistically significant between the study groups (Table 1). Mean homocysteine level was 16.96 ± 7.84 mmol/L in exfoliative subjects (group II and III combined) and 15.07 ± 5.68 mmol/L in those of nonexfoliative (group I and IV combined) subjects, but this difference again did not achieve statistical significance (P = 0.168).
TABLE 1. Comparison of Prothrombotic Risk Factors of Patients With POAG, XFG, XFS, and Control Subjects
Homocysteine (mmol/L) Vitamin B12 (pg/mL) Folate (ng/mL) Activated partial thromboplastin time (aPTT) (s) Antithrombin III activity (%) Protein C activity (%) Free protein S activity (%) Activated protein C resistance (APCR) (s)
Group I (POAG) (n = 25)
Group II (XFG) (n = 25)
Group III (XFS) (n = 25)
Group IV (Control) (n = 25)
P
14.3 ± 4.1 375.7 ± 154.1 10.5 ± 3.3 28.8 ± 1.7
16.8 ± 7.7 394.3 ± 151.3 10.2 ± 2.9 29.3 ± 3.7
17.1 ± 8.1 308.3 ± 96.8 9.5 ± 3.4 29.3 ± 3.8
15.9 ± 6.9 367.3 ± 184.4 9.6 ± 2.9 29.4 ± 3.2
0.514 0.820 0.596 0.948
112.1 ± 11.9 127.9 ± 30.1 115.6 ± 28.9 139.3 ± 24.3
110.1 ± 13.3 120.5 ± 36.0 121.6 ± 36.4 142.1 ± 35.7
110.3 ± 12.9 113.8 ± 33.3 114.4 ± 31.2 135.3 ± 34.2
108.5 ± 19.6 115.4 ± 24.7 116.4 ± 31.4 133.4 ± 33.2
0.714 0.380 0.849 0.597
POAG indicates primary open-angle glaucoma; XFG, exfoliation glaucoma; XFS, exfoliation syndrome.
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J Glaucoma
Volume 25, Number 2, February 2016
Hereditary Thrombophilic Factors in Glaucoma
TABLE 2. Genotype Frequencies of MTHFR C677T, Prothrombin G20210A, and Factor V Leiden Mutations in POAG, XFG, XFS, and Healthy Control Subjects
No. Patients (%) Type of Mutations MTHFR C677T mutation Heterozygous (CT) Homozygous (TT) Prothrombin G20210A mutation Heterozygous (GA) Homozygous (AA) Factor V Leiden mutation Heterozygous (GA) Homozygous (AA)
Group I (POAG)
Group II (XFG)
Group III (XFS)
Group IV (Control)
8 (32) 3 (12)
10 (40) 4 (16)
12 (48) 3 (12)
11(44) 4 (16)
0 (0) 0 (0)
4 (16) 0 (0)
1 (4) 0 (0)
1 (4) 0 (0)
3 (12) 0 (0)
4 (16) 1 (4)
3 (12) 0 (0)
5 (20) 1 (4)
MTHFR indicates methylenetetrahydrofolate reductase; POAG, primary open-angle glaucoma; XFG, exfoliation glaucoma; XFS, exfoliation syndrome.
With respect to the MTHFR C677T mutation, there were 3 homozygous and 8 heterozygous carriers (44%) in group I, 4 homozygous and 10 heterozygous carriers (56%) in group II, 4 homozygous and 12 heterozygous carriers (64%) in group III, and 3 homozygous and 11 heterozygous carriers (56%) in group IV (Table 2). Although the MTHFR C677T mutation appeared to be more prevalent in groups II and III compared with groups I and IV, this difference did not reach statistical significance (P = 0.588). Serum homocysteine levels in the TT phenotype (19.5 ± 9.9 mmol/L) were significantly elevated when compared with TC (15.9 ± 7.2 mmol/L) and CC (14.9 ± 5.0 mmol/L) phenotypes. With respect to the factor V Leiden mutation, there were no homozygous and 3 heterozygous carriers in group I, 1 homozygous and 4 heterozygous carriers in group II, no homozygous and 4 heterozygous carriers in group III, and 3 homozygous and 5 heterozygous carriers in group IV. There were no homozygous carriers of prothrombin G20210A mutation within the 100 subjects enrolled; however, there were 4 heterozygous carriers in group II and 1 heterozygous carrier in groups III and IV. (Table 2) We were not able to test statistical significance related to prothrombin G20210A and factor V Leiden mutations because of lack of mutations in some group.
DISCUSSION In the present study, certain coagulation factors, homocysteine, MTHFR C677T mutation, prothrombin G20210A mutation, and factor V Leiden mutations were all similar in study groups. All the above mentioned parameters are thought to induce a hypercoagulable state and thus may lead to a reduction of ocular blood flow. Controversial results about some of their potential role in the pathogenesis of glaucoma have been previously reported.17–20 To the best of our knowledge, this is the first assessment of the potential role of the prothrombin G20210A and factor V Leiden mutation in the pathogenesis of glaucoma. Our results were unable to find any evidence that related to the presence of elevated prothrombotic factors and mutations in glaucomatous subjects. Antithrombin, protein S, and protein C inhibit specific steps of the coagulation cascade, thereby reducing thrombin generation and the conversion of fibrinogen into fibrin. In our study, antithrombin III activity, protein C activity, and free protein S activity were similar in all groups. Copyright
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MTHFR enzyme catalyzes the reduction of 5,10methylenetetrahydrofolate to 5-methyltetrahydrofolate, the methyl donor of homocysteine to methionine conversion. C677T single-nucleotide polymorphism (SNP) is one of the most extensively studied genetic polymorphism of MTHFR gene.21 The C677T SNP results in a missense mutation leading to substitution of valine for alanine at position 222 of MTHFR enzyme, causing the synthesis of a thermolabile enzyme with a reduction in activity (55% to 65% loss in enzyme activity in individuals who are homozygous for mutation-TT, 25% loss in activity in the heterozygotes-CT when compared with CC homozygous normal individuals).22 Reduced activity of MTHFR leads to deficient production of 5-methyltetrahydrofolate, the cofactor required for the methylation of homocysteine to methionine by methionine synthase. Lack of appropriate methylation of homocysteine results in hyperhomocysteinemia.23 Serum homocysteine levels also have been known to be dependent on some other factors such as folate and vitamin B12, which were also similar in all study groups in the present study.24 To date there are conflicting results regarding the association of MTHFR polymorphism with glaucoma. Several studies have reported an association of C677T polymorphism with glaucoma, whereas this polymorphism appears to play no role in the development of glaucoma based on some others.25 Bleich et al26 found an increased level of plasma homocysteine in white glaucoma patients with the C677T polymorphism. Homocysteine can induce vascular injuries,27 alterations in the extracellular matrix,28 and neuronal cell death by inducing apoptosis or excitotoxicity.12,13 More recently, hyperhomocysteinemia has been shown to be involved in the structural remodeling of connective tissues.29 Previous evidence has suggested restructuring of the sclera in the acute primary angle-closure glaucoma.30 Micheal et al14 proposed that the change in the activity of MTHFR results in hyperhomocysteinemia and in turn induce differential expression of matrix metalloproteases leading to tissue remodeling, and thereby contributing to the pathogenesis of primary angle-closure glaucoma. Altintas¸ et al31 demonstrated that elevation in the levels of homocysteine was associated with exfoliation glaucoma. Ju¨nemann et al6 reported increased frequency of C677T polymorphism of MTHFR in POAG but not in XFG patients. They stated that the MTHFR C677T variant leading to moderate hyperhomocysteinemia may play a role in the pathogenesis of POAG acting as a genetic risk factor.
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Factor V is procofactor for coagulation cascade. Proteolysis of factor V by factor Xa yields factor Va, which in turn is cleaved by APC at Arg506. The G-A transition at nucleotide 1691 in factor V Leiden mutation leads to loss of Arg506, reduces the factor Va proteolysis, thereby diminishes its anticoagulant activity and accelerates thrombosis.32 The factor V Leiden mutation is reported to be present in 5% of whites and conferring APC resistance and a 3- to 7-fold increased risk of venous thrombosis.33 To the best of our knowledge, this is the first study to investigate an association of factor V Leiden mutation with various types of glaucoma. Heterozygous factor V Leiden mutation (GA) was present in 15%, and homozygous mutation (AA) was present in 2% of our study population. But the frequency of these mutations was not high enough to test statistical difference between study groups. Prothrombin, the precursor of thrombin, is a procoagulant enzyme which acts by activating platelets and enhancing fibrin, factors Va, VIIIa, and XIIIa formation. The prothrombin G20210A is a point mutation leading to altered prothrombin mRNA stability and hence higher prothrombin levels, leading to increased risk of thrombosis.15 The prothrombin G20210A mutation is reported to be present in 2% of whites and conferring a 2- to 3-fold increased risk of venous thrombosis.15 Van Cott et al7 demonstrated prothrombin gene mutation G20210A in ocular thrombosis, but to the best of our knowledge, this is the first study to investigate an association of this mutation with various types of glaucoma. Heterozygous prothrombin G20210A mutation (GA) was present in 6% of our study population, but homozygous mutation (AA) was present in none. The frequency of these mutations was not high enough to test statistical difference between study groups. Micheal et al14 investigated the ethnic origin of the patients and its correlation with the disease, and reported different genotype frequencies in different ethnic groups. This study should be viewed in context of this limitation, and it should be kept in mind that ethnic differences may be the main governing factor and prevalence of all aforementioned polymorphisms may vary in different ethnic populations and also in different types of glaucoma. One important limitation of this study is the lack of a priori sample size calculation. In this study, post hoc calculation of the statistical power rather than a calculation of the sample size was performed due to the paucity of published data regarding the thrombophilic factors on glaucomatous subjects. Although the relatively small sizes of the groups in this study may be a limitation, the present study is unique in that several systemic confounding factors of patients have been taken into consideration in the design of the study and an extensive array of thrombotic factors have been simultaneously evaluated in glaucomatous subjects. To the best of our knowledge, this is the first study to investigate the association of prothrombin G20210A and factor V Leiden mutations with various types of glaucoma. In conclusion, our results suggest that there is no statistically significant difference between the prothrombotic inherited risk factors of glaucomatous and nonglaucomatous subjects. The relationship between hereditary thrombophilic factors and glaucoma warrants further study. The extent to which prothrombotic risk factors participate in the pathogenesis of glaucoma is still an enigma.
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ACKNOWLEDGMENT The authors thank Umut Arslan from Hacettepe University Department of Biostatistics, for the statistical analysis.
REFERENCES 1. Van Buskirk EM, Cioffi GA. Glaucomatous optic neuropathy. Am J Ophthalmol. 1992;113:447–452. 2. VanVeldhuisen PC, Ederer F, Gaasterland DE, et al. The Advanced Glaucoma Intervention Study (AGIS): 7. The relationship between control of intraocular pressure and visual field deterioration. The AGIS Investigators. Am J Ophthalmol. 2000;130:429–440. 3. Grieshaber MC, Flammer J. Blood flow in glaucoma. Curr Opin Ophthalmol. 2005;16:79–83. 4. Flammer J, Orgu¨l S, Costa VP, et al. The impact of ocular blood flow in glaucoma. Prog Retin Eye Res. 2002;21:359–393. 5. Grieshaber MC, Mozaffarieh M, Flammer J. What is the link between vascular dysregulation and glaucoma? Surv Ophthalmol. 2007;52(suppl 2):S144–S154. 6. Ju¨nemann AG, von Ahsen N, Reulbach U, et al. C677T variant in the methylentetrahydrofolate reductase gene is a genetic risk factor for primary open-angle glaucoma. Am J Ophthalmol. 2005;139:721–723. 7. Van Cott EM, Laposata M, Hartnett ME. Prothrombin gene mutation G20210A, homocysteine, antiphospholipid antibodies and other hypercoagulable states in ocular thrombosis. Blood Coagul Fibrinolysis. 2004;15:393–397. 8. Wright AD, Martin N, Dodson PM. Homocysteine, folates, and the eye. Eye (Lond). 2008;22:989–993. 9. Leibovitch I, Kurtz S, Shemesh G, et al. Hyperhomocystinemia in pseudoexfoliation glaucoma. J Glaucoma. 2003;12:36–39. 10. Turac¸li ME, Tekeli O, Ozdemir F, et al. Methylenetetrahydrofolate reductase 677 C-T and homocysteine levels in Turkish patients with pseudoexfoliation. Clin Exp Ophthalmol. 2005;33:505–508. 11. Bleich S, Roedl J, Von Ahsen N, et al. Elevated homocysteine levels in aqueous humor of patients with pseudoexfoliation glaucoma. Am J Ophthalmol. 2004;138:162–164. 12. Lipton SA, Kim WK, Choi YB, et al. Neurotoxicity associated with dual actions of homocysteine at the N-methyl-D-aspartate receptor. Proc Natl Acad Sci U S A. 1997;94:5923–5928. 13. Moore P, El-sherbeny A, Roon P, et al. Apoptotic cell death in the mouse retinal ganglion cell layer is induced in vivo by the excitatory amino acid homocysteine. Exp Eye Res. 2001;73: 45–57. 14. Micheal S, Qamar R, Akhtar F, et al. MTHFR gene C677T and A1298C polymorphisms and homocysteine levels in primary open angle and primary closed angle glaucoma. Mol Vis. 2009;15:2268–2278. 15. Poort SR, Rosendaal FR, Reitsma PH, et al. A common genetic variation in the 30 -untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood. 1996; 88:3698–3703. 16. Bertina RM, Koeleman BP, Koster T, et al. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature. 1994;369:64–67. 17. Michael S, Qamar R, Akhtar F, et al. C677T polymorphism in the methylenetetrahydrofolate reductase gene is associated with primary closed angle glaucoma. Mol Vis. 2008;14: 661–665. 18. Zetterberg M, Tasa G, Palme´r MS, et al. Methylenetetrahydrofolate reductase genetic polymorphisms in patients with primary open-angle glaucoma. Ophthalmic Genet. 2007;28: 47–50. 19. Mossbock G, Weger M, Faschinger C, et al. Methylenetetrahydrofolate reductase (MTHFR) 677C > T polymorphism and open angle glaucoma. Mol Vis. 2006;12:356–359.
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20. Fingert JH, Kwon YH, Moore PA, et al. The C677T variant in the methylenetetrahydrofolate reductase gene is not associated with disease in cohorts of pseudoexfoliation glaucoma and primary open-angle glaucoma patients from Iowa. Ophthalmic Genet. 2006;27:39–41. 21. Yamada K, Chen Z, Rozen R, et al. Effects of common polymorphisms on the properties of recombinant human methylenetetrahydrofolate reductase. Proc Natl Acad Sci U S A. 2001;98:14853–14858. 22. Ueland PM, Hustad S, Schneede J, et al. Biological and clinical implications of the MTHFR C677T polymorphism. Trends Pharmacol Sci. 2001;22:195–201. 23. Weisberg IS, Jacques PF, Selhub J, et al. The 1298A– > C polymorphism in methylenetetrahydrofolate reductase (MTHFR): in vitro expression and association with homocysteine. Atherosclerosis. 2001;156:409–415. 24. Sen SK, Pukazhvanthen P, Abraham R. Plasma homocysteine, folate and vitamin B(12) levels in senile cataract. Indian J Clin Biochem. 2008;23:255–257. 25. Xu F, Zhao X, Zeng SM, et al. Homocysteine, B vitamins, methylenetetrahydrofolate reductase gene, and risk of primary open-angle glaucoma: a meta-analysis. Ophthalmology. 2012; 119:2493–2499. 26. Bleich S, Ju¨nemann A, von Ahsen N, et al. Homocysteine and risk of open-angle glaucoma. J Neural Transm. 2002;109: 1499–1504.
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27. McCully KS. Chemical pathology of homocysteine. I. Atherogenesis. Ann Clin Lab Sci. 1993;23:477–493. 28. Tyagi SC. Homocysteine redox receptor and regulation of extracellular matrix components in vascular cells. Am J Physiol. 1998;274:C396–C405. 29. Mujumdar VS, Tummalapalli CM, Aru GM, et al. Mechanism of constrictive vascular remodeling by homocysteine: role of PPAR. Am J Physiol Cell Physiol. 2002;282:C1009–C1015. 30. Wang IJ, Chiang TH, Shih YF, et al. The association of single nucleotide polymorphisms in the MMP-9 genes with susceptibility to acute primary angle closure glaucoma in Taiwanese patients. Mol Vis. 2006;12:1223–1232. 31. Altintas¸ O, Maral H, Yu¨ksel N, et al. Homocysteine and nitric oxide levels in plasma of patients with pseudoexfoliation syndrome, pseudoexfoliation glaucoma, and primary openangle glaucoma. Graefes Arch Clin Exp Ophthalmol. 2005; 243:677–683. 32. Almawi WY, Ameen G, Tamim H, et al. Factor V G1691A, prothrombin G20210A, and methylenetetrahydrofolate reductase [MTHFR] C677T gene polymorphism in angiographically documented coronary artery disease. J Thromb Thrombolysis. 2004;17:199–205. 33. Ridker PM, Hennekens CH, Lindpaintner K, et al. Mutation in the gene coding for coagulation factor V and the risk of myocardial infarction, stroke, and venous thrombosis in apparently healthy men. N Engl J Med. 1995;332:912–917.
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Hereditary Thrombophilic Factors in Glaucoma Mehmet A. Sekeroglu, MD,* Murat Irkec, MD,w Mehmet C. Mocan, MD,w and Mehmet Orhan, MDw
Purpose: To evaluate the hereditary thrombophilic factors in patients with primary open-angle glaucoma, exfoliative glaucoma, and exfoliation syndrome and to compare their results with those of healthy control subjects. Materials and Methods: The study included 75 patients [25 patients with primary open-angle glaucoma (group I), 25 patients with exfoliative glaucoma (group II), and 25 patients with exfoliation syndrome (group III)] and 25 healthy control subjects (group IV). Well-known hereditary thrombophilic factors including methylenetetrahydrofolate reductase (MTHFR) gene C677T mutation, prothrombin G20210A mutation, factor V Leiden mutation, activated protein C resistance, protein S, protein C, and antithrombin III activities, and homocysteine levels were measured in venous blood samples of all subjects. Results: Fifty-one males and 49 females were included in the study. The mean age of the patients was 67.8 ± 8.7 years (range, 46 to 87 y). There was no statistically significant difference with regard to the mean age (P = 0.057) and distribution of sex (P = 0.391) between the study groups. The difference of homocysteine, folate, vitamin B12, antithrombin III activity, protein C activity, free protein S activity, and activated protein C resistance were not statistically significant; and the number of subjects with MTHFR C677T, prothrombin G20210A, and factor V Leiden mutations were similar between the study groups. Conclusion: Our results suggest that there is no significant difference between the prothrombotic inherited risk factors of glaucomatous and nonglaucomatous subjects. Key Words: exfoliation, glaucoma, homocysteine, thrombosis
(J Glaucoma 2016;25:203–207)
G
laucoma, which is one of the leading causes of irreversible blindness in the world, is a progressive multifactorial optic neuropathy characterized by optic nerve head excavation and retinal ganglion cell loss with corresponding visual field damages.1 Although intraocular pressure (IOP) is assumed to be the most significant risk factor for the development of glaucoma,2 the exact mechanism(s) leading to glaucomatous damage have not been clarified yet. In addition to increased IOP, vascular, immunologic, and neurotoxic factors have also been proposed in the pathogenesis of glaucoma. The pathogenic Received for publication October 26, 2013; accepted August 22, 2014. From the *Ulucanlar Eye Training and Research Hospital; and wDepartment of Ophthalmology, Hacettepe University Faculty of Medicine, Ankara, Turkey. The study was conducted in Department of Ophthalmology, Hacettepe University Faculty of Medicine, Ankara, Turkey. Disclosure: The authors declare no conflict of interest. Reprints: Mehmet A. Sekeroglu, MD, Ulucanlar Eye Training and Research Hospital, 06230, Ankara, Turkey (e-mail: msekeroglu@ yahoo.com). Copyright r 2014 Wolters Kluwer Health, Inc. All rights reserved. DOI: 10.1097/IJG.0000000000000148
J Glaucoma
Volume 25, Number 2, February 2016
mechanisms other than increased IOP, especially the vascular factors, gained greater importance in recent years.3–5 Impaired microcirculation of the optic nerve head because of the vascular risk factors may be due to anatomic or functional abnormalities of these vessels. Although the underlying reasons for impaired microcirculation of the optic nerve head in patients with glaucoma are not clear, one possible pathogenetic mechanism may involve abnormalities in prothrombotic inherited risk factors such as the presence of methylenetetrahydrofolate reductase (MTHFR) C677T mutation,6 prothrombin G20210A mutation, factor V Leiden mutation,7 activated protein C resistance (APCR), deficiencies in protein S, protein C, and antithrombin III activities, and elevated homocysteine levels,8 all of which may lead to a greater tendency for thrombosis and may produce a hypercoagulable state which in turn may result in impaired microcirculation of the optic nerve head. Mild hyperhomocysteinemia has been shown to be associated with primary open-angle glaucoma (POAG) and secondary open-angle glaucoma due to exfoliation syndrome (XFS).9,10 Furthermore, significant elevated homocysteine levels have been observed in the aqueous humor of XFG.11 Homocysteine induces apoptotic cell death in retinal ganglion cells by overstimulation of N-methyl-Daspartate receptors and caspase-3 activation.12,13 The purpose of the present study was to evaluate the most common hereditary thrombophilic factors that produce a hypercoagulable state in patients with POAG, exfoliative glaucoma (XFG), and XFS and to compare these parameters with those of healthy control subjects.
MATERIALS AND METHODS The study was designed as a prospective, case-control study undertaken at a single university-based hospital. Informed consent was obtained from all patients and the study was carried out with approval from the Institutional Review Board/Ethics Committee. A total of 100 consecutive subjects [25 POAG (group I), 25 XFG (group II), 25 XFS (group III), and 25 healthy control subjects (group IV)] were recruited between January 2007 and December 2008 for this study. Only patients who fulfilled the selection criteria and gave written informed consent in line with the Declaration of Helsinki were included in the study. All patients underwent complete ophthalmologic examination including best-corrected Snellen visual acuity testing, slit-lamp examination, Goldmann applanation tonometry, ultrasound corneal pachymetry, gonioscopic evaluation, dilated fundus examination using a 90 D lens, and visual field evaluation with the 30-2 SITA-Standard algorithm (Humphrey Visual Field Analyzer; Humphrey Instruments Inc., San Leandro, CA). A detailed medical history was also obtained. www.glaucomajournal.com |
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The diagnosis of POAG was established upon the presence of consistently elevated IOP > 21 mm Hg, normal anterior segment and gonioscopic findings, characteristic optic disc changes, and glaucomatous visual field defects. The diagnosis of XFS was made on slit-lamp examination following mydriasis, as well as gonioscopic evaluation, and included the presence of exfoliation material on the pupillary margin and the anterior lens capsule and trabecular hyperpigmentation. The diagnosis of XFG was made when anterior segment findings of XFS accompanied an IOP > 21 mm Hg without treatment, typical optic nerve head changes, and visual field defects. Twenty-five consecutive subjects aged above 45 years with no history of ocular disease except for refractive errors or presbyopia, an IOPr21 mm Hg, a normal optic disc appearance, and who had no visual field defects were included as controls. The criteria for exclusion included ocular diseases besides refraction disorders, POAG, XFG, XFS, and a history of past ocular surgery; systemic diseases such as diabetes mellitus, cardiovascular disease, dyslipidemia, renal failure, malignancy, autoimmune diseases, hematological diseases, chronic obstructive pulmonary disease, uncontrolled arterial hypertension, and transient ischemic attack or stroke; those who use some medications (such as fibrates, carbamazepine, phenytoin, methotrexate, and trimethoprim), vitamin supplements, or alcohol that may affect plasma homocysteine levels; and those with a history of central nervous system disease (multiple sclerosis, optic nerve glioma, optic neuritis, optic nerve sheath meningioma, and intracranial tumors) that might have interfered with visual field testing. After at least 8 hours of fasting, fresh peripheral whole blood samples of all subjects were collected from the antecubital vein at the same time interval of the day (9 to 11 AM). Fasting blood glucose, liver and renal function tests, serum triglycerides and cholesterol, vitamin B12, folate, hemoglobin, and hematocrit levels were determined in all subjects. Hematological tests were carried out using STA analyser (Diagnostica Stago Asnieres, France) and included activated partial thromboplastin time-(aPTT) (STAC.K.Prest), antithrombin III activity-AT III (chromogenic substrate assay; STA-AT III), protein C activity (synthetic chromogenic substrate method; STA-Protein C), and free protein S activity (immunoturbidimetric method; STALiatest Free Protein S) assays. Serum homocysteine levels were determined with a fluorescence polarization immunoassay (AXSYM-Homocysteine, AXSYM System; Abbott, Wiesbaden, Germany).
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For the analysis of MTHFR C677T, prothrombin G20210A, and factor V Leiden mutations, genomic DNA was extracted from 4 mL of EDTA anticoagulated blood by conventional phenol-chloroform method. Polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) was used for genotype analysis as mentioned in detail elsewhere.14–16 Statistical analyses were performed using SPSS version 15.0 (SPSS Inc., Chicago, IL) software. One-way analysis of variance (ANOVA) was used to compare quantitative data between multiple groups when a normal distribution was found and Kruskal-Wallis 1-way ANOVA on ranks was used if data were not normally distributed. Kruskal-Wallis Z-value test was used for all pairwise multiple comparisons. Student unpaired t test or the Mann-Whitney U test was used to compare quantitative data and w2 analysis was used for qualitative data. For all evaluations, P < 0.05 was considered significant. Post hoc calculation of statistical power was performed using NCSS-PASS software (NCSS, UT).
RESULTS A total of 100 subjects [25 patients with POAG (group I), 25 patients with XFG (group II), 25 patients with XFS (group III), and 25 healthy control subjects (group IV)] were included in the study. The mean age of the patients was 67.8 ± 8.7 years (range, 46 to 87 y). Of the 100 subjects, 51 were males and 49 were females. The mean age of group I was 64.4 ± 8.5 years (10 males, 15 females), group II was 68.9 ± 9.1 years (16 males, 9 females), group III was 70.8 ± 7.0 years (13 males, 12 females), and group IV was 67.3 ± 9.2 years (12 males, 13 females). There was no statistically significant difference with regard to the mean age (P = 0.057) and distribution of sex (P = 0.391) between the study groups. The complete blood counts including platelets were within normal limits in all subjects. None of the participants had elevated prothrombin levels and aPTT. The difference of homocysteine, folate, vitamin B12, aPTT, antithrombin III activity, protein C activity, free protein S activity, and APCR levels were not statistically significant between the study groups (Table 1). Mean homocysteine level was 16.96 ± 7.84 mmol/L in exfoliative subjects (group II and III combined) and 15.07 ± 5.68 mmol/L in those of nonexfoliative (group I and IV combined) subjects, but this difference again did not achieve statistical significance (P = 0.168).
TABLE 1. Comparison of Prothrombotic Risk Factors of Patients With POAG, XFG, XFS, and Control Subjects
Homocysteine (mmol/L) Vitamin B12 (pg/mL) Folate (ng/mL) Activated partial thromboplastin time (aPTT) (s) Antithrombin III activity (%) Protein C activity (%) Free protein S activity (%) Activated protein C resistance (APCR) (s)
Group I (POAG) (n = 25)
Group II (XFG) (n = 25)
Group III (XFS) (n = 25)
Group IV (Control) (n = 25)
P
14.3 ± 4.1 375.7 ± 154.1 10.5 ± 3.3 28.8 ± 1.7
16.8 ± 7.7 394.3 ± 151.3 10.2 ± 2.9 29.3 ± 3.7
17.1 ± 8.1 308.3 ± 96.8 9.5 ± 3.4 29.3 ± 3.8
15.9 ± 6.9 367.3 ± 184.4 9.6 ± 2.9 29.4 ± 3.2
0.514 0.820 0.596 0.948
112.1 ± 11.9 127.9 ± 30.1 115.6 ± 28.9 139.3 ± 24.3
110.1 ± 13.3 120.5 ± 36.0 121.6 ± 36.4 142.1 ± 35.7
110.3 ± 12.9 113.8 ± 33.3 114.4 ± 31.2 135.3 ± 34.2
108.5 ± 19.6 115.4 ± 24.7 116.4 ± 31.4 133.4 ± 33.2
0.714 0.380 0.849 0.597
POAG indicates primary open-angle glaucoma; XFG, exfoliation glaucoma; XFS, exfoliation syndrome.
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J Glaucoma
Volume 25, Number 2, February 2016
Hereditary Thrombophilic Factors in Glaucoma
TABLE 2. Genotype Frequencies of MTHFR C677T, Prothrombin G20210A, and Factor V Leiden Mutations in POAG, XFG, XFS, and Healthy Control Subjects
No. Patients (%) Type of Mutations MTHFR C677T mutation Heterozygous (CT) Homozygous (TT) Prothrombin G20210A mutation Heterozygous (GA) Homozygous (AA) Factor V Leiden mutation Heterozygous (GA) Homozygous (AA)
Group I (POAG)
Group II (XFG)
Group III (XFS)
Group IV (Control)
8 (32) 3 (12)
10 (40) 4 (16)
12 (48) 3 (12)
11(44) 4 (16)
0 (0) 0 (0)
4 (16) 0 (0)
1 (4) 0 (0)
1 (4) 0 (0)
3 (12) 0 (0)
4 (16) 1 (4)
3 (12) 0 (0)
5 (20) 1 (4)
MTHFR indicates methylenetetrahydrofolate reductase; POAG, primary open-angle glaucoma; XFG, exfoliation glaucoma; XFS, exfoliation syndrome.
With respect to the MTHFR C677T mutation, there were 3 homozygous and 8 heterozygous carriers (44%) in group I, 4 homozygous and 10 heterozygous carriers (56%) in group II, 4 homozygous and 12 heterozygous carriers (64%) in group III, and 3 homozygous and 11 heterozygous carriers (56%) in group IV (Table 2). Although the MTHFR C677T mutation appeared to be more prevalent in groups II and III compared with groups I and IV, this difference did not reach statistical significance (P = 0.588). Serum homocysteine levels in the TT phenotype (19.5 ± 9.9 mmol/L) were significantly elevated when compared with TC (15.9 ± 7.2 mmol/L) and CC (14.9 ± 5.0 mmol/L) phenotypes. With respect to the factor V Leiden mutation, there were no homozygous and 3 heterozygous carriers in group I, 1 homozygous and 4 heterozygous carriers in group II, no homozygous and 4 heterozygous carriers in group III, and 3 homozygous and 5 heterozygous carriers in group IV. There were no homozygous carriers of prothrombin G20210A mutation within the 100 subjects enrolled; however, there were 4 heterozygous carriers in group II and 1 heterozygous carrier in groups III and IV. (Table 2) We were not able to test statistical significance related to prothrombin G20210A and factor V Leiden mutations because of lack of mutations in some group.
DISCUSSION In the present study, certain coagulation factors, homocysteine, MTHFR C677T mutation, prothrombin G20210A mutation, and factor V Leiden mutations were all similar in study groups. All the above mentioned parameters are thought to induce a hypercoagulable state and thus may lead to a reduction of ocular blood flow. Controversial results about some of their potential role in the pathogenesis of glaucoma have been previously reported.17–20 To the best of our knowledge, this is the first assessment of the potential role of the prothrombin G20210A and factor V Leiden mutation in the pathogenesis of glaucoma. Our results were unable to find any evidence that related to the presence of elevated prothrombotic factors and mutations in glaucomatous subjects. Antithrombin, protein S, and protein C inhibit specific steps of the coagulation cascade, thereby reducing thrombin generation and the conversion of fibrinogen into fibrin. In our study, antithrombin III activity, protein C activity, and free protein S activity were similar in all groups. Copyright
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MTHFR enzyme catalyzes the reduction of 5,10methylenetetrahydrofolate to 5-methyltetrahydrofolate, the methyl donor of homocysteine to methionine conversion. C677T single-nucleotide polymorphism (SNP) is one of the most extensively studied genetic polymorphism of MTHFR gene.21 The C677T SNP results in a missense mutation leading to substitution of valine for alanine at position 222 of MTHFR enzyme, causing the synthesis of a thermolabile enzyme with a reduction in activity (55% to 65% loss in enzyme activity in individuals who are homozygous for mutation-TT, 25% loss in activity in the heterozygotes-CT when compared with CC homozygous normal individuals).22 Reduced activity of MTHFR leads to deficient production of 5-methyltetrahydrofolate, the cofactor required for the methylation of homocysteine to methionine by methionine synthase. Lack of appropriate methylation of homocysteine results in hyperhomocysteinemia.23 Serum homocysteine levels also have been known to be dependent on some other factors such as folate and vitamin B12, which were also similar in all study groups in the present study.24 To date there are conflicting results regarding the association of MTHFR polymorphism with glaucoma. Several studies have reported an association of C677T polymorphism with glaucoma, whereas this polymorphism appears to play no role in the development of glaucoma based on some others.25 Bleich et al26 found an increased level of plasma homocysteine in white glaucoma patients with the C677T polymorphism. Homocysteine can induce vascular injuries,27 alterations in the extracellular matrix,28 and neuronal cell death by inducing apoptosis or excitotoxicity.12,13 More recently, hyperhomocysteinemia has been shown to be involved in the structural remodeling of connective tissues.29 Previous evidence has suggested restructuring of the sclera in the acute primary angle-closure glaucoma.30 Micheal et al14 proposed that the change in the activity of MTHFR results in hyperhomocysteinemia and in turn induce differential expression of matrix metalloproteases leading to tissue remodeling, and thereby contributing to the pathogenesis of primary angle-closure glaucoma. Altintas¸ et al31 demonstrated that elevation in the levels of homocysteine was associated with exfoliation glaucoma. Ju¨nemann et al6 reported increased frequency of C677T polymorphism of MTHFR in POAG but not in XFG patients. They stated that the MTHFR C677T variant leading to moderate hyperhomocysteinemia may play a role in the pathogenesis of POAG acting as a genetic risk factor.
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Factor V is procofactor for coagulation cascade. Proteolysis of factor V by factor Xa yields factor Va, which in turn is cleaved by APC at Arg506. The G-A transition at nucleotide 1691 in factor V Leiden mutation leads to loss of Arg506, reduces the factor Va proteolysis, thereby diminishes its anticoagulant activity and accelerates thrombosis.32 The factor V Leiden mutation is reported to be present in 5% of whites and conferring APC resistance and a 3- to 7-fold increased risk of venous thrombosis.33 To the best of our knowledge, this is the first study to investigate an association of factor V Leiden mutation with various types of glaucoma. Heterozygous factor V Leiden mutation (GA) was present in 15%, and homozygous mutation (AA) was present in 2% of our study population. But the frequency of these mutations was not high enough to test statistical difference between study groups. Prothrombin, the precursor of thrombin, is a procoagulant enzyme which acts by activating platelets and enhancing fibrin, factors Va, VIIIa, and XIIIa formation. The prothrombin G20210A is a point mutation leading to altered prothrombin mRNA stability and hence higher prothrombin levels, leading to increased risk of thrombosis.15 The prothrombin G20210A mutation is reported to be present in 2% of whites and conferring a 2- to 3-fold increased risk of venous thrombosis.15 Van Cott et al7 demonstrated prothrombin gene mutation G20210A in ocular thrombosis, but to the best of our knowledge, this is the first study to investigate an association of this mutation with various types of glaucoma. Heterozygous prothrombin G20210A mutation (GA) was present in 6% of our study population, but homozygous mutation (AA) was present in none. The frequency of these mutations was not high enough to test statistical difference between study groups. Micheal et al14 investigated the ethnic origin of the patients and its correlation with the disease, and reported different genotype frequencies in different ethnic groups. This study should be viewed in context of this limitation, and it should be kept in mind that ethnic differences may be the main governing factor and prevalence of all aforementioned polymorphisms may vary in different ethnic populations and also in different types of glaucoma. One important limitation of this study is the lack of a priori sample size calculation. In this study, post hoc calculation of the statistical power rather than a calculation of the sample size was performed due to the paucity of published data regarding the thrombophilic factors on glaucomatous subjects. Although the relatively small sizes of the groups in this study may be a limitation, the present study is unique in that several systemic confounding factors of patients have been taken into consideration in the design of the study and an extensive array of thrombotic factors have been simultaneously evaluated in glaucomatous subjects. To the best of our knowledge, this is the first study to investigate the association of prothrombin G20210A and factor V Leiden mutations with various types of glaucoma. In conclusion, our results suggest that there is no statistically significant difference between the prothrombotic inherited risk factors of glaucomatous and nonglaucomatous subjects. The relationship between hereditary thrombophilic factors and glaucoma warrants further study. The extent to which prothrombotic risk factors participate in the pathogenesis of glaucoma is still an enigma.
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ACKNOWLEDGMENT The authors thank Umut Arslan from Hacettepe University Department of Biostatistics, for the statistical analysis.
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20. Fingert JH, Kwon YH, Moore PA, et al. The C677T variant in the methylenetetrahydrofolate reductase gene is not associated with disease in cohorts of pseudoexfoliation glaucoma and primary open-angle glaucoma patients from Iowa. Ophthalmic Genet. 2006;27:39–41. 21. Yamada K, Chen Z, Rozen R, et al. Effects of common polymorphisms on the properties of recombinant human methylenetetrahydrofolate reductase. Proc Natl Acad Sci U S A. 2001;98:14853–14858. 22. Ueland PM, Hustad S, Schneede J, et al. Biological and clinical implications of the MTHFR C677T polymorphism. Trends Pharmacol Sci. 2001;22:195–201. 23. Weisberg IS, Jacques PF, Selhub J, et al. The 1298A– > C polymorphism in methylenetetrahydrofolate reductase (MTHFR): in vitro expression and association with homocysteine. Atherosclerosis. 2001;156:409–415. 24. Sen SK, Pukazhvanthen P, Abraham R. Plasma homocysteine, folate and vitamin B(12) levels in senile cataract. Indian J Clin Biochem. 2008;23:255–257. 25. Xu F, Zhao X, Zeng SM, et al. Homocysteine, B vitamins, methylenetetrahydrofolate reductase gene, and risk of primary open-angle glaucoma: a meta-analysis. Ophthalmology. 2012; 119:2493–2499. 26. Bleich S, Ju¨nemann A, von Ahsen N, et al. Homocysteine and risk of open-angle glaucoma. J Neural Transm. 2002;109: 1499–1504.
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27. McCully KS. Chemical pathology of homocysteine. I. Atherogenesis. Ann Clin Lab Sci. 1993;23:477–493. 28. Tyagi SC. Homocysteine redox receptor and regulation of extracellular matrix components in vascular cells. Am J Physiol. 1998;274:C396–C405. 29. Mujumdar VS, Tummalapalli CM, Aru GM, et al. Mechanism of constrictive vascular remodeling by homocysteine: role of PPAR. Am J Physiol Cell Physiol. 2002;282:C1009–C1015. 30. Wang IJ, Chiang TH, Shih YF, et al. The association of single nucleotide polymorphisms in the MMP-9 genes with susceptibility to acute primary angle closure glaucoma in Taiwanese patients. Mol Vis. 2006;12:1223–1232. 31. Altintas¸ O, Maral H, Yu¨ksel N, et al. Homocysteine and nitric oxide levels in plasma of patients with pseudoexfoliation syndrome, pseudoexfoliation glaucoma, and primary openangle glaucoma. Graefes Arch Clin Exp Ophthalmol. 2005; 243:677–683. 32. Almawi WY, Ameen G, Tamim H, et al. Factor V G1691A, prothrombin G20210A, and methylenetetrahydrofolate reductase [MTHFR] C677T gene polymorphism in angiographically documented coronary artery disease. J Thromb Thrombolysis. 2004;17:199–205. 33. Ridker PM, Hennekens CH, Lindpaintner K, et al. Mutation in the gene coding for coagulation factor V and the risk of myocardial infarction, stroke, and venous thrombosis in apparently healthy men. N Engl J Med. 1995;332:912–917.
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