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G20210A Prothrombin Mutation and Critical Limb Ischaemia in Patients with Peripheral Arterial Disease
Eur J Vasc Endovasc Surg (2009) 38, 113e117
G20210A Prothrombin Mutation and Critical Limb Ischaemia in Patients with Peripheral Arterial Disease M. Sartori*, E. Favaretto, C. Legnani, M. Cini, E. Conti, C. Pili, G. Palareti Department of Angiology and Blood Coagulation, S. Orsola-Malpighi, University Hospital, 40138 Bologna, Italy Submitted 20 October 2008; accepted 24 February 2009 Available online 7 April 2009
KEYWORDS Peripheral arterial disease; Lower limb artery disease; Arterial thrombosis; Thrombophilic risk factor; G20210A prothrombin; R506Q FV Leiden
Abstract Objectives: To assess the possible association between inherited thrombophilic alterations and the severity of peripheral arterial disease (PAD). Design: A case-control study. Methods: We evaluated the presence of G20210A prothrombin (FII) and R506Q FV Leiden mutations, antithrombin, protein C and S deficiencies in 176 patients with PAD at Fontaine’s stage II and in 106 patients with critical limb ischaemia (Fontaine’s stage III/IV) consecutively referred to our unit. As control group, we studied 209 apparently healthy subjects. Results: The prevalence of G20210A prothrombin mutation was similar in PAD patients and controls (odds ratio (OR): 1.361; 95% confidence interval (CI): 0.552e3.355; p Z 0.503 after adjustment for age, sex, smoking and presence of diabetes), but was significantly higher in patients with Fontaine’s stage III/IV vs. those with stage II and controls (10.4% vs. 3.4% vs. 4.3%; p Z 0.02, respectively). According to a logistic multivariate model that included all patients with PAD, the presence of the FII G20210A mutation (OR: 4.621; 95% CI: 1.548e 13.789; p Z 0.006) was associated with critical limb ischaemia after adjustment for age, sex, smoking, presence of diabetes and the use of platelet aggregation inhibitors. The prevalence of the other thrombophilic alterations was not different in patients with Fontaine’s stage III/IV, in patients with stage II and in controls. Conclusion: These hypothesis-generating data suggest that the FII 20210A allele may be considered as a genetic marker predisposing critical ischaemia in patients with PAD, justifying larger longitudinal studies. ª 2009 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. M. Sartori, U.O. Angiologia e Malattie della Coagulazione ‘Marino Golinelli’, Azienda Ospedaliera di Bologna, Policlinico Sant’ Orsola-Malpighi, Pad. 2, Via Albertoni, 15, 40138 Bologna, Italy. Tel.: þ39 051 636 2482; fax: þ39 051 636 2517. E-mail address: [email protected] (M. Sartori).
Atherosclerotic peripheral arterial disease (PAD) is usually a benign condition characterised by arterial stenoses and occlusions in the peripheral arterial bed of the lower limbs. PAD can be asymptomatic or symptomatic with different
1078-5884/$36 ª 2009 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ejvs.2009.02.014
114 symptoms depending on the progression of the disease. Intermittent claudication is usually the first symptom and remains stable in 75% of patients, while a progression of the disease leading to clinical deterioration (severe claudication or skin lesions) occurs in the remainder.1 Black race, current smoking, diabetes, hypertension, hypercholesterolaemia and low kidney function are the well-known risk factors for PAD.2 Diabetes and current smoking are related to PAD progression as well.3 The role of haemostatic factor levels and inflammatory markers, such as D-dimer and fibrinogen, has been investigated in PAD.4 However, the true relationship between thrombophilic abnormalities and atherosclerosis progression remains a matter of controversy. A recent meta-analysis showed a modest but significant relationship between inherited thrombophilic alterations and thrombotic arterial events.5 Thrombosis may play an important role in the pathogenesis of atherosclerotic complications. Genetic polymorphisms of haemostatic factors such as G1691A factor V (FV Leiden) and G20210A prothrombin (FII) may be involved in arterial thrombosis and may interact with traditional cardiovascular risk factors, leading to clinical deterioration, such as critical limb ischaemia. Gerdes et al. showed that FII G20210A mutation was associated with increased femoral intimal thickness and with the occurrence of acute ischaemic events in patients with clinically manifest atherosclerotic disease.6 In our hypotheses, the presence of FII G20210A and FV Leiden mutations might be a limited risk factor for PAD, but when arterial stenoses in the peripheral arterial bed are present such mutations may be aggravating factors. Given the large number of patients affected by PAD and the importance of detecting the patients at risk of clinical deterioration, the aim of the present study was to assess the association of FII G20210A and FV Leiden mutations with the severity of PAD symptoms.
Methods Study population We investigated 282 Caucasian patients (females/males: 101/181) consecutively admitted to our Angiology Unit (from January 2005 to September 2007) for lower limb artery disease. Subjects were diagnosed with PAD if they had clinical PAD symptoms (intermittent claudication, rest pain or gangrene), ankleebrachial index (ABI) <0.9 and significant stenoses of leg arteries confirmed by echo-Doppler sonography. Exclusion criteria were: PAD from non-atherosclerotic causes (cardio-embolic disease, thromboangiitis obliterans, vasculitis or congenital), presence of neoplasia and severe dementia. Fontaine stages were assigned as follows: class IIa, mild claudication with a walking distance of more than 200 m; class IIb, severe claudication with a walking distance of less than 200 m; class III, rest pain and class IV, gangrene. Stage III and IV were grouped as critical ischaemia.7 Stage IIa and IIb were grouped as claudication. For analysis, patients were divided into two groups: patients with intermittent claudication (n Z 176) and patients with critical limb ischaemia (n Z 106).
M. Sartori et al. As a control group, we selected 209 Caucasian subjects (females/males: 87/122) referred to our outpatient service, in whom the disease had been excluded on the basis of physical examination and normal ABI. The patients enrolled in the present study were considered to have hypertension if diagnosed as hypertensives according to the guidelines of the European Society of Hypertension.8 Hyperlipidaemia was defined according to the third report of the National Cholesterol Education Program.9 Subjects were considered diabetic if they used glucose-lowering medication or had fasting blood glucose levels of 126 mg dl1 or higher. Obesity was defined as a body mass index equal to or higher than 30 kg m2.10 The study, which complied with the Declaration of Helsinki, was approved by the local Ethics Committee and informed consent was obtained from all participants.
Vascular investigations All PAD patients underwent bilateral ultrasonography assessment of lower limb arteries and ABI determination. Ultrasonography investigation was carried out with an EnVisor C HD instrument (Philips Medical System S.p.A, Monza, Italy), with a high-resolution broadband width linear array transducer L 12-5 MHz, according to standardised methods.11,12 ABI was calculated according to the American Heart Association recommendations13 and values >0.9 were considered as normal. All investigations were performed by a team of trained vascular physicians or technicians. When clinically required, patients underwent a conventional contrast angiography scanning via the femoral or axillary route.
Blood sampling and thrombophilia screening Blood was collected from the antecubital vein into 0.109 mmol l1 trisodium citrate. Plasma was prepared by centrifugation for 20 min at 2000 g at room temperature while platelet-poor plasma was snap frozen and stored at 70 C. The screening for inherited thrombophilia included the following tests: antithrombin, protein C, protein S, activated protein C resistance (APCR) with DNA analysis for G1691A FV mutation in all cases with APCR normalised ratio <0.80, and DNA analysis for FII G20210A mutation. All tests were performed using standard methods as previously described.14 Genotyping of G1691A FV and G20210A FII polymorphisms was performed successfully in 100% of subjects.
Statistical analysis Analysis was carried out using the SPSS software package (version 15.0; SPSS Inc., Chicago, IL, USA). Relationships between variables were assessed using Pearson correlation for continuous variables and chi-square or Fisher’s exact test for categorical variables. Multivariate analysis of variance with Tukey correction for multiple comparisons was used to compare means among groups. HardyeWeinberg equilibrium was assessed by the chi-square test with 1 of freedom. Odds ratios were calculated with multivariate logistic regression analysis including the following
Peripheral Arterial Disease and Thrombophilic Mutations variables: G20210A FII mutation, G1691A FV mutation, sex, age, smoking habit, the presence of diabetes and the use of platelet aggregation inhibitors. The significance level was set at a Z 0.05. Results are given as means SEM. The power of the study was calculated with the ‘genetic power calculator’ available on the web at http://statgen. iop.kcl.ac.uk/gpc/.15 The power of the study was 0.91 (a Z 0.05) when the relative risk for PAD of 20210A carriers was set >3.5, according to the studies of Reny et al.16 and Sofi et al.,17 and the frequency of the A allele was set at 0.022. The power of the study increased to 0.95 if the odds ratio (OR) for critical ischaemia in patients carrying the 20210A allele was set at 4.1, as already reported by Reny et al.16
Results The characteristics and traditional atherosclerosis risk factors in patients and controls are summarised in Table 1. Among PAD patients, 176 had intermittent claudication and 106 had critical limb ischaemia (Table 1). Sex distribution, prevalence of hypertension, hyperlipidaemia and obesity were similar among control subjects, patients with claudication and patients with critical limb ischaemia (Table 1). The mean age of patients with critical ischaemia was higher than that of control subjects (72.1 0.9 vs. 68.5 0.4 years; p Z 0.01) and of patients with claudication (72.1 0.9 vs. 68.6 0.7 years; p Z 0.01). As expected, an increased prevalence of smoking in patients with claudication and critical limb ischaemia was observed, whereas the prevalence of hypertension, hyperlipidaemia and obesity was similar among the three groups (Table 1). Prevalence of diabetes was higher in patients with critical ischaemia than in patients with claudication and was higher in patients with claudication vs. control subjects (Table 1). The distributions of genotypes of G1691A FV mutation in the control group, in patients with claudication and in patients with critical ischaemia were in HardyeWeinberg equilibrium (in controls: frequency of allele G, 0.975; frequency of allele A, 0.025; chi-square Z 0.13; p Z 0.72; in patients with claudication: frequency of allele G, 0.980; frequency of allele A, 0.020; chi-square Z 0.07; p Z 0.79; in patients with critical ischaemia: frequency of allele G, 0.976; frequency of allele A, 0.024; chi-square Z 0.06; p Z 0.81). No difference was found between cases and Table 1
a
c
controls regarding the distribution of factor V G1691A genotypes as tested by the chi-square test (Table 2) and, accordingly, no significant OR (assessed by logistic regression analysis) for the A allele was observed. The distributions of genotypes of FII G20210A mutation in the control group, in patients with claudication and in patients with critical ischaemia were also in HardyeWeinberg equilibrium (in controls: frequency of allele G, 0.978; frequency of allele A, 0.022; chi-square Z 0.1; p Z 0.75; in patients with claudication: frequency of allele G, 0.983; frequency of allele A, 0.017; chi-square Z 0.05; p Z 0.82; in patients with critical ischaemia: frequency of allele G, 0.943; frequency of allele A, 0.057; chi-square Z 1.44; p Z 0.23). The prevalence of FII G20210A mutation was similar in PAD patients and controls (OR: 1.426; 95% confidence interval (CI): 0.623e3.263; p Z 0.424 and OR: 1.361; 95% CI: 0.552e3.355; p Z 0.503 after adjustment for age, sex, smoking, and presence of diabetes). As shown in Table 2, there was a significant difference among control subjects, patients with claudication and patients with critical ischaemia with respect to the distribution of FII G20210A genotypes (the presence of the A allele was associated with critical ischaemia, with an OR vs. claudication of 3.281; CI: 1.176e9.152; p Z 0.021). According to a logistic multivariate model, including only patients with PAD, the presence of the FII G20210A mutation (95% CI: 1.548e13.789; p Z 0.006), diabetes, (95% CI: 1.362e4.151; p Z 0.03) and age (95% CI: 1.009e1.071; p Z 0.01) were associated with critical ischaemia, whereas sex distribution, smoking and the presence of FV G1691A mutation were not (Table 3). Use of platelet aggregation inhibitors had a negative association (Table 3). No patient in either the PAD group or control group had deficiencies in antithrombin, protein C or protein S levels.
Discussion Our study shows that the FII G20210A mutation may be associated with more disabling stages of PAD while no association was found between PAD and G1691A FV mutation. FV Leiden and FII G20210A mutations are widely accepted as risk factors for venous thrombo-embolism,18 but their role in arterial thrombotic disease and atherosclerosis is controversial.
Characteristics of the study population.
Age (years) Male/female Obesity (%) Hypertension (%) Diabetes (%) Hyperlipidaemia (%) Smoking (%) b
115
p-valuea
Controls (n Z 209)
Patients with peripheral artery disease Claudication (n Z 176)
Critical ischaemia (n Z 106)
68.5 0.4 122/87 1.9 36.4 3.3 30.1 10.0
68.6 0.7 114/62 3.4 42.0 17.0 38.1 33.5
72.1 0.9b, 67/39 2.8 34.9 35.8b, c 27.4 33.0c
c
0.001 0.410 0.651 0.389 0.0001 0.117 0.0001
The statistical analysis was performed by multivariate analysis of variance for age, and the chi-square test for the other variables. p Z 0.01 vs. claudication group. p Z 0.01 vs. control group.
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M. Sartori et al.
Table 2 Allele and genotype frequencies of prothrombin gene G20210A and R506Q FV Leiden mutation in Italian patients with peripheral artery disease (PAD) and controls.
Factor V G1691A Alleles A G Genotypes AA GA GG
p-valuea
Controls (n Z 209)
Patients with PAD Claudication (n Z 176)
Critical ischaemia (n Z 106)
10/418 (2.5) 408/418 (97.5)
7/352 (2.0) 345/352 (98.0)
5/212 (2.4) 207/212 (97.6)
0.922
0/209 10/209 (4.8) 199/209 (95.2)
0/176 7/176 (4.0) 169/176 (96.0)
0/106 5/106 (4.7) 101/106 (95.3)
0.922
6/352 (1.7) 346/352 (98.3)
12/212 (5.7) 200/212 (94.3)
0.01
0/176 6/176 (3.4) 170/176 (93.2)
1/106 (0.9) 10/106 (9.5) 95/106 (89.6)
0.029
Prothrombin G20210A Alleles A 9/418 (2.2) G 409/418 (97.8) Genotypes AA 0/209 GA 9/209 (4.3) GG 200/209 (95.7)
Data presented as number with variable/total number possible (%). a Comparisons between the control group, claudication group, and critical limb ischaemia group were assessed by the chi-square test.
The FII 20210A allele has already been found to be associated with atherosclerosis progression in carotid and femoral arteries in men.6 Reny et al.16 demonstrated that the FII G20210A mutation was significantly associated with the increased risk of PAD, a risk which markedly increased with smoking. FII 20210A allele was also associated with critical ischaemia in a multivariate analysis.16 An Italian case-control study17 showed a significantly higher prevalence of FII G20210A mutation in PAD patients than in controls, but no correlation was found with PAD stage. Conversely, three case-control studies showed no association between FII G20210A mutation and PAD.19e21 The negative results of such studies can be ascribed to different characteristics and risk profiles of the study population. In the study of Renner et al.,19 the mean age of the controls was 16 years younger than that of the patients. Moreover, a low frequency of patients with more advanced stages of PAD may have masked the effect of the FII G20210A mutation. In a study by Mueller et al.,20 only 17% of patients
Table 3 Logistic multivariate model for critical limb ischaemia among patients with peripheral artery disease (n Z 282). Age Sex (female vs. male) Smoking Presence of diabetes Use of aggregation inhibitors FII 20210A allele FV 1691A allele
OR
CI 95%
p
1.040 0.911 1.452 1.480 0.379 4.621 0.862
1.009e1.071 0.524e1.581 0.819e2.574 1.362e4.1514 0.222e0.649 1.548e13.789 0.240e3.094
0.01 0.70 0.202 0.03 0.001 0.006 0.820
with PAD had critical ischaemia, a number that was even lower in patients included in the study of Abukishe et al.21 We were able to enrol a larger number of patients with critical ischaemia because we enrolled patients who were older than those in the series of Reny al.16 and of Abukishe et al.21 In the present study, we found that the prevalence of FII G20210A mutation was not increased in patients with initial PAD stages, suggesting a limited role of the FII G20210A mutation in PAD aetiology. Our data suggest, albeit with the limitations of the small sample size, that G20210A FII mutation may become an aggravating factor, possibly increasing the risk of critical limb ischaemia development when PAD is already present. In fact, a prospective study has shown that the 20210A allele is associated with atherosclerosis progression in the carotid and femoral arteries in patients with established atherosclerotic disease.6 Heterozygosity for the 20210A allele is associated with a 25% increase in circulating prothrombin levels.22 The association of the mutation in the prothrombin gene with excessive thrombin generation and activation of coagulation can lead to a ‘prothrombotic’ state.22 Arterial thrombosis results from endovascular injury and, to a lesser extent, alterations in haemostatic equilibrium.23 Endothelial cell injury with the elaboration of proinflammatory mediators stimulates the process of arterial thrombosis.23 Although this is most often the result of atherosclerotic disease, variations in the activity of coagulation proteins may alter the effectiveness of thrombus formation, leading to PAD progression. As seen in larger studies,3 we confirmed that age and diabetes were associated to more severe stages of PAD. It is well established that smoking, the ratio of total to highdensity lipoprotein (HDL) cholesterol, lipoprotein (a), highsensitivity C-reactive protein and diabetes are related to PAD progression.3
Peripheral Arterial Disease and Thrombophilic Mutations In line with previous studies,16,17,19,20 the prevalence of G1691A FV mutation was similar in patients with claudication or more severe stages of PAD and in controls. It should be noted that in a recent meta-analysis the association between FV Leiden mutation and arterial ischaemic events was modest with an OR of 1.21.5 Given the low number of patients enrolled in the present study, we cannot exclude with any certainty an association between G1691A FV and PAD. However, previous case-control studies exploring thrombophilic risk factors in PAD did not reveal any association.16,17,19,20 No deficiencies in antithrombin, protein C or S levels were found in our series. Given the low prevalence of natural inhibitor deficiencies, our study cannot exclude the possibility that such risk factors may be involved in PAD progression. At present, there are no convincing data that deficiencies of protein C, protein S or antithrombin are associated with an increased risk of arterial thrombosis,23 but the evaluation of these associations is complicated by the relative infrequency of such defects in the general population. Limitations of the present study include the crosssectional design, the small sample size, and the possible presence of confounders such as the higher age of patients with critical ischaemia. Follow-up studies are necessary to confirm the association of 20210A FII allele and PAD progression. Nevertheless, our hypothesis-generating data suggest that the 20210A FII allele may be considered as a genetic marker predisposing to critical ischaemia in patients with established PAD, and a closer follow-up may be useful in patients with PAD carrying the FII G20210A mutation. In our series, FII G20210A mutation was present in 10% of PAD patients with chronic critical limb ischaemia. In conclusion, FII G20210A mutation may be associated with more disabling stages of PAD, though longitudinal studies will better clarify if this mutation is a genetic marker which predicts an individual’s predisposition for PAD progression.
Conflict of Interest None.
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References 19 1 Kannel WB, Shurtleff D. The natural history of arteriosclerosis obliterans. Cardiovasc Clin 1971;3:37e52. 2 Selvin E, Erlinger TP. Prevalence of and risk factors for peripheral arterial disease in the United States: results from the National Health and Nutrition Examination Survey, 1999e2000. Circulation 2004;110:738e43. 3 Aboyans V, Criqui MH, Denenberg JO, Knoke JD, Ridker PM, Fronek A. Risk factors for progression of peripheral arterial disease in large and small vessels. Circulation 2006;113:2623e9. 4 McDermott MM, Green D, Greenland P, Liu K, Criqui MH, Chan C, et al. Relation of levels of hemostatic factors and inflammatory markers to the ankle brachial index. Am J Cardiol 2003;92:194e9. 5 Kim RJ, Becker RC. Association between factor V Leiden, prothrombin G20210A, and methylenetetrahydrofolate reductase C677T mutations and events of the arterial circulatory system: a meta-analysis of published studies. Am Heart J 2003; 146:948e57. 6 Gerdes VE, ten Cate H, de Groot E, Kwa VI, Prins MH, Reitsma PH, et al. Amsterdam Vascular Medicine Group. Arterial wall thickness and the risk of recurrent ischemic events in carriers of the
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prothrombin G20210A mutation with clinical manifestations of atherosclerosis. Atherosclerosis 2002;163:135e40. Santilli JD, Santilli SM. Chronic critical limb ischemia: diagnosis, treatment and prognosis. Am Fam Physician 1999;59:1899e908. 1999 World Health OrganizationeInternational Society of Hypertension. Guidelines for the management of hypertension. J Hypertens 1999;17:151e83. Third report of the National Cholesterol Education Program (NCEP) expert panel on detection, evaluation and treatment of high blood cholesterol in adults (Adult Treatment Panel III). Final report. Circulation 2002;106:3143e421. Sartori M, Ceolotto G, Dorigatti F, Mos L, Santonastaso M, Bratti P, et al. HARVEST Group. RGS2 C1114G polymorphism and body weight gain in hypertensive patients. Metabolism 2008;57:421e7. Ebrahim S, Papacosta O, Whincup P, Wannamethee G, Walker M, Nicolaides AN, et al. Carotid plaque, intima media thickness, cardiovascular risk factors, and prevalent cardiovascular disease in men and women: the British Regional Heart Study. Stroke 1999;30:841e50. Favaretto E, Pili C, Amato A, Conti E, Losinno F, Rossi C, et al. Analysis of agreement between Duplex ultrasound scanning and arteriography in patients with lower limb artery disease. J Cardiovasc Med 2007;8:337e41. Greenland P, Abrams J, Aurigemma GP, Bond MG, Clark LT, Criqui MH, et al. Prevention conference V: beyond secondary prevention: identifying the high-risk patient for primary prevention: non-invasive tests of atherosclerotic burden. Writing Group III. Circulation 2000;101:16e22. Legnani C, Palareti G, Guazzaloca G, Cosmi B, Lunghi B, Bernardi F, et al. Venous thromboembolism in young women; role of thrombophilic mutations and oral contraceptive use. Eur Heart J 2002;23:984e90. Purcell S, Cherny SS, Sham PC. Genetic power calculator: design of linkage and association genetic mapping studies of complex traits. Bioinformatics 2003;19:149e50. Reny JL, Alhenc-Gelas M, Fontana P, Bissery A, Julia PL, Fiessinger JN, et al. The factor II G20210A gene polymorphism, but not factor V Arg506Gln, is associated with peripheral arterial disease: results of a case-control study. J Thromb Haemost 2004;2:1334e40. Sofi F, Lari B, Rogolino A, Marcucci R, Pratesi G, Dorigo W, et al. Thrombophilic risk factors for symptomatic peripheral arterial disease. J Vasc Surg 2005;41:255e60. Cohn DM, Roshani S, Middeldorp S. Thrombophilia and venous thromboembolism: implications for testing. Semin Thromb Hemost 2007;33:573e81. Renner W, Ko ¨ppel H, Brodmann M, Pabst E, Schallmoser K, Toplak H, et al. Factor II G20210A and factor V G1691A gene mutations and peripheral arterial occlusive disease. Thromb Haemost 2000;83:20e2. Mueller T, Marschon R, Dieplinger B, Haidinger D, Gegenhuber A, Poelz W, et al. Factor V Leiden, prothrombin G20210A, and methylenetetrahydrofolate reductase C677T mutations are not associated with chronic limb ischemia: the Linz peripheral arterial disease (LIPAD) study. J Vasc Surg 2005; 41:808e15. Abukishe A, Brandt M, Hedderich J, Hirt S, Lentz S, Scha ¨fer H, et al. Mutation in factor II and factor V gene in patients with peripheral arterial occlusive disease. Hamostaseologie 2006; 26:197e200. Franco RF, Trip MD, ten Cate H, van den Ende A, Prins MH, Kastelein JJ, et al. The 20210 G/A mutation in the 30 -untranslated region of the prothrombin gene and the risk for arterial thrombotic disease. Br J Haematol 1999;104:50e4. Feinbloom D, Bauer KA. Assessment of hemostatic risk factors in predicting arterial thrombotic events. Arterioscler Thromb Vasc Biol 2005;25:2043e53.
G20210A Prothrombin Mutation and Critical Limb Ischaemia in Patients with Peripheral Arterial Disease M. Sartori*, E. Favaretto, C. Legnani, M. Cini, E. Conti, C. Pili, G. Palareti Department of Angiology and Blood Coagulation, S. Orsola-Malpighi, University Hospital, 40138 Bologna, Italy Submitted 20 October 2008; accepted 24 February 2009 Available online 7 April 2009
KEYWORDS Peripheral arterial disease; Lower limb artery disease; Arterial thrombosis; Thrombophilic risk factor; G20210A prothrombin; R506Q FV Leiden
Abstract Objectives: To assess the possible association between inherited thrombophilic alterations and the severity of peripheral arterial disease (PAD). Design: A case-control study. Methods: We evaluated the presence of G20210A prothrombin (FII) and R506Q FV Leiden mutations, antithrombin, protein C and S deficiencies in 176 patients with PAD at Fontaine’s stage II and in 106 patients with critical limb ischaemia (Fontaine’s stage III/IV) consecutively referred to our unit. As control group, we studied 209 apparently healthy subjects. Results: The prevalence of G20210A prothrombin mutation was similar in PAD patients and controls (odds ratio (OR): 1.361; 95% confidence interval (CI): 0.552e3.355; p Z 0.503 after adjustment for age, sex, smoking and presence of diabetes), but was significantly higher in patients with Fontaine’s stage III/IV vs. those with stage II and controls (10.4% vs. 3.4% vs. 4.3%; p Z 0.02, respectively). According to a logistic multivariate model that included all patients with PAD, the presence of the FII G20210A mutation (OR: 4.621; 95% CI: 1.548e 13.789; p Z 0.006) was associated with critical limb ischaemia after adjustment for age, sex, smoking, presence of diabetes and the use of platelet aggregation inhibitors. The prevalence of the other thrombophilic alterations was not different in patients with Fontaine’s stage III/IV, in patients with stage II and in controls. Conclusion: These hypothesis-generating data suggest that the FII 20210A allele may be considered as a genetic marker predisposing critical ischaemia in patients with PAD, justifying larger longitudinal studies. ª 2009 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. M. Sartori, U.O. Angiologia e Malattie della Coagulazione ‘Marino Golinelli’, Azienda Ospedaliera di Bologna, Policlinico Sant’ Orsola-Malpighi, Pad. 2, Via Albertoni, 15, 40138 Bologna, Italy. Tel.: þ39 051 636 2482; fax: þ39 051 636 2517. E-mail address: [email protected] (M. Sartori).
Atherosclerotic peripheral arterial disease (PAD) is usually a benign condition characterised by arterial stenoses and occlusions in the peripheral arterial bed of the lower limbs. PAD can be asymptomatic or symptomatic with different
1078-5884/$36 ª 2009 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ejvs.2009.02.014
114 symptoms depending on the progression of the disease. Intermittent claudication is usually the first symptom and remains stable in 75% of patients, while a progression of the disease leading to clinical deterioration (severe claudication or skin lesions) occurs in the remainder.1 Black race, current smoking, diabetes, hypertension, hypercholesterolaemia and low kidney function are the well-known risk factors for PAD.2 Diabetes and current smoking are related to PAD progression as well.3 The role of haemostatic factor levels and inflammatory markers, such as D-dimer and fibrinogen, has been investigated in PAD.4 However, the true relationship between thrombophilic abnormalities and atherosclerosis progression remains a matter of controversy. A recent meta-analysis showed a modest but significant relationship between inherited thrombophilic alterations and thrombotic arterial events.5 Thrombosis may play an important role in the pathogenesis of atherosclerotic complications. Genetic polymorphisms of haemostatic factors such as G1691A factor V (FV Leiden) and G20210A prothrombin (FII) may be involved in arterial thrombosis and may interact with traditional cardiovascular risk factors, leading to clinical deterioration, such as critical limb ischaemia. Gerdes et al. showed that FII G20210A mutation was associated with increased femoral intimal thickness and with the occurrence of acute ischaemic events in patients with clinically manifest atherosclerotic disease.6 In our hypotheses, the presence of FII G20210A and FV Leiden mutations might be a limited risk factor for PAD, but when arterial stenoses in the peripheral arterial bed are present such mutations may be aggravating factors. Given the large number of patients affected by PAD and the importance of detecting the patients at risk of clinical deterioration, the aim of the present study was to assess the association of FII G20210A and FV Leiden mutations with the severity of PAD symptoms.
Methods Study population We investigated 282 Caucasian patients (females/males: 101/181) consecutively admitted to our Angiology Unit (from January 2005 to September 2007) for lower limb artery disease. Subjects were diagnosed with PAD if they had clinical PAD symptoms (intermittent claudication, rest pain or gangrene), ankleebrachial index (ABI) <0.9 and significant stenoses of leg arteries confirmed by echo-Doppler sonography. Exclusion criteria were: PAD from non-atherosclerotic causes (cardio-embolic disease, thromboangiitis obliterans, vasculitis or congenital), presence of neoplasia and severe dementia. Fontaine stages were assigned as follows: class IIa, mild claudication with a walking distance of more than 200 m; class IIb, severe claudication with a walking distance of less than 200 m; class III, rest pain and class IV, gangrene. Stage III and IV were grouped as critical ischaemia.7 Stage IIa and IIb were grouped as claudication. For analysis, patients were divided into two groups: patients with intermittent claudication (n Z 176) and patients with critical limb ischaemia (n Z 106).
M. Sartori et al. As a control group, we selected 209 Caucasian subjects (females/males: 87/122) referred to our outpatient service, in whom the disease had been excluded on the basis of physical examination and normal ABI. The patients enrolled in the present study were considered to have hypertension if diagnosed as hypertensives according to the guidelines of the European Society of Hypertension.8 Hyperlipidaemia was defined according to the third report of the National Cholesterol Education Program.9 Subjects were considered diabetic if they used glucose-lowering medication or had fasting blood glucose levels of 126 mg dl1 or higher. Obesity was defined as a body mass index equal to or higher than 30 kg m2.10 The study, which complied with the Declaration of Helsinki, was approved by the local Ethics Committee and informed consent was obtained from all participants.
Vascular investigations All PAD patients underwent bilateral ultrasonography assessment of lower limb arteries and ABI determination. Ultrasonography investigation was carried out with an EnVisor C HD instrument (Philips Medical System S.p.A, Monza, Italy), with a high-resolution broadband width linear array transducer L 12-5 MHz, according to standardised methods.11,12 ABI was calculated according to the American Heart Association recommendations13 and values >0.9 were considered as normal. All investigations were performed by a team of trained vascular physicians or technicians. When clinically required, patients underwent a conventional contrast angiography scanning via the femoral or axillary route.
Blood sampling and thrombophilia screening Blood was collected from the antecubital vein into 0.109 mmol l1 trisodium citrate. Plasma was prepared by centrifugation for 20 min at 2000 g at room temperature while platelet-poor plasma was snap frozen and stored at 70 C. The screening for inherited thrombophilia included the following tests: antithrombin, protein C, protein S, activated protein C resistance (APCR) with DNA analysis for G1691A FV mutation in all cases with APCR normalised ratio <0.80, and DNA analysis for FII G20210A mutation. All tests were performed using standard methods as previously described.14 Genotyping of G1691A FV and G20210A FII polymorphisms was performed successfully in 100% of subjects.
Statistical analysis Analysis was carried out using the SPSS software package (version 15.0; SPSS Inc., Chicago, IL, USA). Relationships between variables were assessed using Pearson correlation for continuous variables and chi-square or Fisher’s exact test for categorical variables. Multivariate analysis of variance with Tukey correction for multiple comparisons was used to compare means among groups. HardyeWeinberg equilibrium was assessed by the chi-square test with 1 of freedom. Odds ratios were calculated with multivariate logistic regression analysis including the following
Peripheral Arterial Disease and Thrombophilic Mutations variables: G20210A FII mutation, G1691A FV mutation, sex, age, smoking habit, the presence of diabetes and the use of platelet aggregation inhibitors. The significance level was set at a Z 0.05. Results are given as means SEM. The power of the study was calculated with the ‘genetic power calculator’ available on the web at http://statgen. iop.kcl.ac.uk/gpc/.15 The power of the study was 0.91 (a Z 0.05) when the relative risk for PAD of 20210A carriers was set >3.5, according to the studies of Reny et al.16 and Sofi et al.,17 and the frequency of the A allele was set at 0.022. The power of the study increased to 0.95 if the odds ratio (OR) for critical ischaemia in patients carrying the 20210A allele was set at 4.1, as already reported by Reny et al.16
Results The characteristics and traditional atherosclerosis risk factors in patients and controls are summarised in Table 1. Among PAD patients, 176 had intermittent claudication and 106 had critical limb ischaemia (Table 1). Sex distribution, prevalence of hypertension, hyperlipidaemia and obesity were similar among control subjects, patients with claudication and patients with critical limb ischaemia (Table 1). The mean age of patients with critical ischaemia was higher than that of control subjects (72.1 0.9 vs. 68.5 0.4 years; p Z 0.01) and of patients with claudication (72.1 0.9 vs. 68.6 0.7 years; p Z 0.01). As expected, an increased prevalence of smoking in patients with claudication and critical limb ischaemia was observed, whereas the prevalence of hypertension, hyperlipidaemia and obesity was similar among the three groups (Table 1). Prevalence of diabetes was higher in patients with critical ischaemia than in patients with claudication and was higher in patients with claudication vs. control subjects (Table 1). The distributions of genotypes of G1691A FV mutation in the control group, in patients with claudication and in patients with critical ischaemia were in HardyeWeinberg equilibrium (in controls: frequency of allele G, 0.975; frequency of allele A, 0.025; chi-square Z 0.13; p Z 0.72; in patients with claudication: frequency of allele G, 0.980; frequency of allele A, 0.020; chi-square Z 0.07; p Z 0.79; in patients with critical ischaemia: frequency of allele G, 0.976; frequency of allele A, 0.024; chi-square Z 0.06; p Z 0.81). No difference was found between cases and Table 1
a
c
controls regarding the distribution of factor V G1691A genotypes as tested by the chi-square test (Table 2) and, accordingly, no significant OR (assessed by logistic regression analysis) for the A allele was observed. The distributions of genotypes of FII G20210A mutation in the control group, in patients with claudication and in patients with critical ischaemia were also in HardyeWeinberg equilibrium (in controls: frequency of allele G, 0.978; frequency of allele A, 0.022; chi-square Z 0.1; p Z 0.75; in patients with claudication: frequency of allele G, 0.983; frequency of allele A, 0.017; chi-square Z 0.05; p Z 0.82; in patients with critical ischaemia: frequency of allele G, 0.943; frequency of allele A, 0.057; chi-square Z 1.44; p Z 0.23). The prevalence of FII G20210A mutation was similar in PAD patients and controls (OR: 1.426; 95% confidence interval (CI): 0.623e3.263; p Z 0.424 and OR: 1.361; 95% CI: 0.552e3.355; p Z 0.503 after adjustment for age, sex, smoking, and presence of diabetes). As shown in Table 2, there was a significant difference among control subjects, patients with claudication and patients with critical ischaemia with respect to the distribution of FII G20210A genotypes (the presence of the A allele was associated with critical ischaemia, with an OR vs. claudication of 3.281; CI: 1.176e9.152; p Z 0.021). According to a logistic multivariate model, including only patients with PAD, the presence of the FII G20210A mutation (95% CI: 1.548e13.789; p Z 0.006), diabetes, (95% CI: 1.362e4.151; p Z 0.03) and age (95% CI: 1.009e1.071; p Z 0.01) were associated with critical ischaemia, whereas sex distribution, smoking and the presence of FV G1691A mutation were not (Table 3). Use of platelet aggregation inhibitors had a negative association (Table 3). No patient in either the PAD group or control group had deficiencies in antithrombin, protein C or protein S levels.
Discussion Our study shows that the FII G20210A mutation may be associated with more disabling stages of PAD while no association was found between PAD and G1691A FV mutation. FV Leiden and FII G20210A mutations are widely accepted as risk factors for venous thrombo-embolism,18 but their role in arterial thrombotic disease and atherosclerosis is controversial.
Characteristics of the study population.
Age (years) Male/female Obesity (%) Hypertension (%) Diabetes (%) Hyperlipidaemia (%) Smoking (%) b
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p-valuea
Controls (n Z 209)
Patients with peripheral artery disease Claudication (n Z 176)
Critical ischaemia (n Z 106)
68.5 0.4 122/87 1.9 36.4 3.3 30.1 10.0
68.6 0.7 114/62 3.4 42.0 17.0 38.1 33.5
72.1 0.9b, 67/39 2.8 34.9 35.8b, c 27.4 33.0c
c
0.001 0.410 0.651 0.389 0.0001 0.117 0.0001
The statistical analysis was performed by multivariate analysis of variance for age, and the chi-square test for the other variables. p Z 0.01 vs. claudication group. p Z 0.01 vs. control group.
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Table 2 Allele and genotype frequencies of prothrombin gene G20210A and R506Q FV Leiden mutation in Italian patients with peripheral artery disease (PAD) and controls.
Factor V G1691A Alleles A G Genotypes AA GA GG
p-valuea
Controls (n Z 209)
Patients with PAD Claudication (n Z 176)
Critical ischaemia (n Z 106)
10/418 (2.5) 408/418 (97.5)
7/352 (2.0) 345/352 (98.0)
5/212 (2.4) 207/212 (97.6)
0.922
0/209 10/209 (4.8) 199/209 (95.2)
0/176 7/176 (4.0) 169/176 (96.0)
0/106 5/106 (4.7) 101/106 (95.3)
0.922
6/352 (1.7) 346/352 (98.3)
12/212 (5.7) 200/212 (94.3)
0.01
0/176 6/176 (3.4) 170/176 (93.2)
1/106 (0.9) 10/106 (9.5) 95/106 (89.6)
0.029
Prothrombin G20210A Alleles A 9/418 (2.2) G 409/418 (97.8) Genotypes AA 0/209 GA 9/209 (4.3) GG 200/209 (95.7)
Data presented as number with variable/total number possible (%). a Comparisons between the control group, claudication group, and critical limb ischaemia group were assessed by the chi-square test.
The FII 20210A allele has already been found to be associated with atherosclerosis progression in carotid and femoral arteries in men.6 Reny et al.16 demonstrated that the FII G20210A mutation was significantly associated with the increased risk of PAD, a risk which markedly increased with smoking. FII 20210A allele was also associated with critical ischaemia in a multivariate analysis.16 An Italian case-control study17 showed a significantly higher prevalence of FII G20210A mutation in PAD patients than in controls, but no correlation was found with PAD stage. Conversely, three case-control studies showed no association between FII G20210A mutation and PAD.19e21 The negative results of such studies can be ascribed to different characteristics and risk profiles of the study population. In the study of Renner et al.,19 the mean age of the controls was 16 years younger than that of the patients. Moreover, a low frequency of patients with more advanced stages of PAD may have masked the effect of the FII G20210A mutation. In a study by Mueller et al.,20 only 17% of patients
Table 3 Logistic multivariate model for critical limb ischaemia among patients with peripheral artery disease (n Z 282). Age Sex (female vs. male) Smoking Presence of diabetes Use of aggregation inhibitors FII 20210A allele FV 1691A allele
OR
CI 95%
p
1.040 0.911 1.452 1.480 0.379 4.621 0.862
1.009e1.071 0.524e1.581 0.819e2.574 1.362e4.1514 0.222e0.649 1.548e13.789 0.240e3.094
0.01 0.70 0.202 0.03 0.001 0.006 0.820
with PAD had critical ischaemia, a number that was even lower in patients included in the study of Abukishe et al.21 We were able to enrol a larger number of patients with critical ischaemia because we enrolled patients who were older than those in the series of Reny al.16 and of Abukishe et al.21 In the present study, we found that the prevalence of FII G20210A mutation was not increased in patients with initial PAD stages, suggesting a limited role of the FII G20210A mutation in PAD aetiology. Our data suggest, albeit with the limitations of the small sample size, that G20210A FII mutation may become an aggravating factor, possibly increasing the risk of critical limb ischaemia development when PAD is already present. In fact, a prospective study has shown that the 20210A allele is associated with atherosclerosis progression in the carotid and femoral arteries in patients with established atherosclerotic disease.6 Heterozygosity for the 20210A allele is associated with a 25% increase in circulating prothrombin levels.22 The association of the mutation in the prothrombin gene with excessive thrombin generation and activation of coagulation can lead to a ‘prothrombotic’ state.22 Arterial thrombosis results from endovascular injury and, to a lesser extent, alterations in haemostatic equilibrium.23 Endothelial cell injury with the elaboration of proinflammatory mediators stimulates the process of arterial thrombosis.23 Although this is most often the result of atherosclerotic disease, variations in the activity of coagulation proteins may alter the effectiveness of thrombus formation, leading to PAD progression. As seen in larger studies,3 we confirmed that age and diabetes were associated to more severe stages of PAD. It is well established that smoking, the ratio of total to highdensity lipoprotein (HDL) cholesterol, lipoprotein (a), highsensitivity C-reactive protein and diabetes are related to PAD progression.3
Peripheral Arterial Disease and Thrombophilic Mutations In line with previous studies,16,17,19,20 the prevalence of G1691A FV mutation was similar in patients with claudication or more severe stages of PAD and in controls. It should be noted that in a recent meta-analysis the association between FV Leiden mutation and arterial ischaemic events was modest with an OR of 1.21.5 Given the low number of patients enrolled in the present study, we cannot exclude with any certainty an association between G1691A FV and PAD. However, previous case-control studies exploring thrombophilic risk factors in PAD did not reveal any association.16,17,19,20 No deficiencies in antithrombin, protein C or S levels were found in our series. Given the low prevalence of natural inhibitor deficiencies, our study cannot exclude the possibility that such risk factors may be involved in PAD progression. At present, there are no convincing data that deficiencies of protein C, protein S or antithrombin are associated with an increased risk of arterial thrombosis,23 but the evaluation of these associations is complicated by the relative infrequency of such defects in the general population. Limitations of the present study include the crosssectional design, the small sample size, and the possible presence of confounders such as the higher age of patients with critical ischaemia. Follow-up studies are necessary to confirm the association of 20210A FII allele and PAD progression. Nevertheless, our hypothesis-generating data suggest that the 20210A FII allele may be considered as a genetic marker predisposing to critical ischaemia in patients with established PAD, and a closer follow-up may be useful in patients with PAD carrying the FII G20210A mutation. In our series, FII G20210A mutation was present in 10% of PAD patients with chronic critical limb ischaemia. In conclusion, FII G20210A mutation may be associated with more disabling stages of PAD, though longitudinal studies will better clarify if this mutation is a genetic marker which predicts an individual’s predisposition for PAD progression.
Conflict of Interest None.
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References 19 1 Kannel WB, Shurtleff D. The natural history of arteriosclerosis obliterans. Cardiovasc Clin 1971;3:37e52. 2 Selvin E, Erlinger TP. Prevalence of and risk factors for peripheral arterial disease in the United States: results from the National Health and Nutrition Examination Survey, 1999e2000. Circulation 2004;110:738e43. 3 Aboyans V, Criqui MH, Denenberg JO, Knoke JD, Ridker PM, Fronek A. Risk factors for progression of peripheral arterial disease in large and small vessels. Circulation 2006;113:2623e9. 4 McDermott MM, Green D, Greenland P, Liu K, Criqui MH, Chan C, et al. Relation of levels of hemostatic factors and inflammatory markers to the ankle brachial index. Am J Cardiol 2003;92:194e9. 5 Kim RJ, Becker RC. Association between factor V Leiden, prothrombin G20210A, and methylenetetrahydrofolate reductase C677T mutations and events of the arterial circulatory system: a meta-analysis of published studies. Am Heart J 2003; 146:948e57. 6 Gerdes VE, ten Cate H, de Groot E, Kwa VI, Prins MH, Reitsma PH, et al. Amsterdam Vascular Medicine Group. Arterial wall thickness and the risk of recurrent ischemic events in carriers of the
20
21
22
23
prothrombin G20210A mutation with clinical manifestations of atherosclerosis. Atherosclerosis 2002;163:135e40. Santilli JD, Santilli SM. Chronic critical limb ischemia: diagnosis, treatment and prognosis. Am Fam Physician 1999;59:1899e908. 1999 World Health OrganizationeInternational Society of Hypertension. Guidelines for the management of hypertension. J Hypertens 1999;17:151e83. Third report of the National Cholesterol Education Program (NCEP) expert panel on detection, evaluation and treatment of high blood cholesterol in adults (Adult Treatment Panel III). Final report. Circulation 2002;106:3143e421. Sartori M, Ceolotto G, Dorigatti F, Mos L, Santonastaso M, Bratti P, et al. HARVEST Group. RGS2 C1114G polymorphism and body weight gain in hypertensive patients. Metabolism 2008;57:421e7. Ebrahim S, Papacosta O, Whincup P, Wannamethee G, Walker M, Nicolaides AN, et al. Carotid plaque, intima media thickness, cardiovascular risk factors, and prevalent cardiovascular disease in men and women: the British Regional Heart Study. Stroke 1999;30:841e50. Favaretto E, Pili C, Amato A, Conti E, Losinno F, Rossi C, et al. Analysis of agreement between Duplex ultrasound scanning and arteriography in patients with lower limb artery disease. J Cardiovasc Med 2007;8:337e41. Greenland P, Abrams J, Aurigemma GP, Bond MG, Clark LT, Criqui MH, et al. Prevention conference V: beyond secondary prevention: identifying the high-risk patient for primary prevention: non-invasive tests of atherosclerotic burden. Writing Group III. Circulation 2000;101:16e22. Legnani C, Palareti G, Guazzaloca G, Cosmi B, Lunghi B, Bernardi F, et al. Venous thromboembolism in young women; role of thrombophilic mutations and oral contraceptive use. Eur Heart J 2002;23:984e90. Purcell S, Cherny SS, Sham PC. Genetic power calculator: design of linkage and association genetic mapping studies of complex traits. Bioinformatics 2003;19:149e50. Reny JL, Alhenc-Gelas M, Fontana P, Bissery A, Julia PL, Fiessinger JN, et al. The factor II G20210A gene polymorphism, but not factor V Arg506Gln, is associated with peripheral arterial disease: results of a case-control study. J Thromb Haemost 2004;2:1334e40. Sofi F, Lari B, Rogolino A, Marcucci R, Pratesi G, Dorigo W, et al. Thrombophilic risk factors for symptomatic peripheral arterial disease. J Vasc Surg 2005;41:255e60. Cohn DM, Roshani S, Middeldorp S. Thrombophilia and venous thromboembolism: implications for testing. Semin Thromb Hemost 2007;33:573e81. Renner W, Ko ¨ppel H, Brodmann M, Pabst E, Schallmoser K, Toplak H, et al. Factor II G20210A and factor V G1691A gene mutations and peripheral arterial occlusive disease. Thromb Haemost 2000;83:20e2. Mueller T, Marschon R, Dieplinger B, Haidinger D, Gegenhuber A, Poelz W, et al. Factor V Leiden, prothrombin G20210A, and methylenetetrahydrofolate reductase C677T mutations are not associated with chronic limb ischemia: the Linz peripheral arterial disease (LIPAD) study. J Vasc Surg 2005; 41:808e15. Abukishe A, Brandt M, Hedderich J, Hirt S, Lentz S, Scha ¨fer H, et al. Mutation in factor II and factor V gene in patients with peripheral arterial occlusive disease. Hamostaseologie 2006; 26:197e200. Franco RF, Trip MD, ten Cate H, van den Ende A, Prins MH, Kastelein JJ, et al. The 20210 G/A mutation in the 30 -untranslated region of the prothrombin gene and the risk for arterial thrombotic disease. Br J Haematol 1999;104:50e4. Feinbloom D, Bauer KA. Assessment of hemostatic risk factors in predicting arterial thrombotic events. Arterioscler Thromb Vasc Biol 2005;25:2043e53.