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Keywords:

  • Behçet's disease;
  • Deep vein thrombosis;
  • Factor V Leiden mutation, Prothrombin G20210A mutation

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Objective

To evaluate the frequency and type of vascular lesions and to study the association of factor V gene G1691A (Leiden) and prothrombin gene G20210A polymorphisms with venous thrombosis in Italian patients with Behçet's disease (BD).

Methods

Included were 118 consecutive Italian BD patients followed over a 3-year period (1997–1999) who satisfied the International Study Group criteria for BD. The control group consisted of 132 healthy Italian blood donors. All BD patients and controls were genotyped by polymerase chain reaction and allele-specific restriction enzyme techniques for factor V Leiden and prothrombin gene G20210A polymorphisms.

Results

Vascular lesions were observed in 37 (31.4%) patients. The 2 most common lesions were subcutaneous thrombophlebitis (10.2%) and deep vein thrombosis (DVT) of the legs (22.8%). No significant demographic and clinical differences between patients with and without DVT were present. The distribution of allele and genotype frequencies of prothrombin gene G20210A and factor V Leiden polymorphisms did not differ significantly between BD patients and healthy controls. The frequencies of carriage rates of prothrombin gene G20210A and factor V Leiden polymorphisms in BD patients with and without DVT were similar. However, the frequency of 20210A allele was significantly higher in BD patients with ocular disease than in those without, particularly in the patients with posterior uveitis/retinal vasculitis.

Conclusions

The frequency and types of vascular lesions in Italian BD patients were similar to those reported in studies from other countries. No association between factor V Leiden mutation and G20210A mutation in the 3′-untranslated region of the prothrombin gene with DVT was found. However, a prothrombin gene G20210A mutation may influence the development and severity of ocular involvement in BD.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Behçet's disease (BD) is a multisystemic inflammatory disease of unknown origin characterized by recurrent oral aphtous ulcers, genital ulcers, uveitis, and skin lesions (1, 2). Vasculitis is the pathologic lesion common to most of the clinical manifestations of BD. Vascular lesions, although not included in the diagnostic criteria for BD by the International Study Group (3), are detected in 10–30% of patients (4). Superficial thrombophlebitis and involvement of the deep veins of the extremities are the most common vascular lesions.

The pathogenesis of thrombosis in BD is unknown, although the contribution of multiple factors is likely. Various abnormalities related to endothelial cell functions have been described in BD (4–9). Endothelial dysfunction resulting from vasculitis could play an important role in the pathogenesis of thrombotic manifestations observed in BD. However, thrombophilia may also play a key role. The factor V Leiden mutation is the most common cause of inherited thrombophilia. A single point mutation 1691G to A in the factor V gene produces a 506Arg to Gln replacement in the cleavage site of the factor V moiety, making it resistant to degradation by activated protein C. This mutation increases the risk of venous thrombosis (10–12). An odds ratio (OR) for venous thrombosis of 6.2 was estimated for factor V Leiden carriers in a large random sample of the Italian population (13). Discordant data have been reported on the association between deep vein thrombosis (DVT) and factor V Leiden mutation in BD (14–20).

A second mutation that is an established inherited cause of thrombophilia is the G20210A mutation in the 3′-untranslated region of the prothrombin gene. This mutation is reported to increase the risk of venous thrombosis by almost 3-fold (21). The 20210A allele is associated with elevated prothrombin levels, itself a risk factor of thrombosis (22). Interestingly, its geographic distribution parallels the prevalence of BD, the highest prevalence being found in southern Europe and the Middle East (23), where the prevalence of BD is higher. The association between this mutation and venous thrombosis in BD has been studied in Turkish and Spanish patients, with discordant data being reported (15, 20, 24).

The aim of this study was to determine whether or not these 2 mutations were associated with an increased risk of venous thrombosis in Italian patients with BD. We also evaluated the frequency and the types of vascular lesions in our series of Italian patients.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Patients

Participants were consecutive BD patients followed at the Rheumatology, Ophthalmology, and Neurology Units in hospitals in Bologna, Ferrara, Milano, Palermo, Potenza, Prato, Reggio Emilia, and Trento during a 3-year period (1997–1999) who satisfied the International Study Group criteria for BD (3). Of the 125 patients that were recruited, 7 were excluded because they were of northern African or Turkish origin. The 118 patients that were white, of Italian descent, and residing in Italy for at least 1 generation were the cohort of patients studied. The control group consisted of 132 white people of Italian descent who were unrelated blood donor volunteers. No ethnic differences were present between patients and controls, and none were of Jewish background. The study was approved by the Ethics Committees of the participating centers and informed consent was obtained from patients and controls before inclusion in the study.

The diagnosis of DVT and subcutaneous thrombophlebitis was based on clinical data and confirmed by ultrasonography or contrast venography.

HLA class I typing

Serologic HLA class I typing was performed by the standard microlymphocytotoxicity technique, using peripheral blood lymphocytes. Of the 118 Italian patients with BD, 94 were typed for HLA–B51. The control group was made up of 130 Italian healthy subjects.

Polymerase chain reaction (PCR) analysis of prothrombin gene G20210A mutation and factor V gene G1691A mutation

Blood for molecular biology analysis was drawn by venipuncture of the antecubital vein in trisodium citrate (129 mmol/liter) 1:9 vol:vol and DNA was extracted with InstaGene whole blood kit (Bio-Rad, Hercules, CA) on fresh or frozen whole blood samples. PCR was performed with AmpliTaq polymerase on a 9600 Thermal Cycler, both from Perkin Elmer (Emeryville, CA), using specific in-house synthesised primers.

For prothrombin gene G20210A, the primers were TCT AGA AAC AGT TGC CTG GC (forward) and ATA GCA CTG GGA GCA TTG AA*GC (reverse), where * indicates a primer specific for the mutated allele. The amplification mixture was as follows: dNTPs 200 mM, forward primer 20 pmole, reverse primer 20 pmole, buffer 1×, Taq DNA polymerase 1.5 U, DNA 10 μl, distilled water 50 μl. Amplification procedure was as follows: first 2 minutes at 94°C, then 30 seconds at 94°C, 30 seconds at 60°C, and 40 seconds at 72°C for 35 cycles, and finally 5 minutes at 72°C. The restriction enzyme digestion mixture was as follows: Hinf I restriction endonuclease 1 U, buffer 1×, bovine serum albumin 2 mg, amplified DNA 17 μl; the mixture was incubated for 2 hours at 37°C.

Electrophoresis was performed on a 4% agarose gel for 1 hour at 85 volts (25).

For factor V Leiden gene G1691A mutation, the primers were GGA ACA ACA CCA TGA TCA GAG CA (forward) and TAG CCA GGA GAC CTA ACA TGT TC (reverse). The amplification mixture was as follows: dNTPs 200 mM, forward primer 20 pmole, reverse primer 20 pmole, buffer 1×, Taq DNA polymerase 1.5 U, DNA 10 μl, distilled water 50 μl. Amplification procedure was as follows: first 2 minutes at 94°C, then 30 seconds at 94°C, 30 seconds at 60°C, and 40 seconds at 72°C for 35 cycles, and finally 5 minutes at 72°C.

The restriction enzyme digestion mixture was as follows: Mnl I restriction endonuclease 1 U, buffer 1×, bovine serum albumin 2 mg, amplified DNA 17 μl; the mixture was incubated for 2 hours at 37°C.

Electrophoresis was performed on a 3% agarose gel for 45 minutes at 85 volts (22).

Statistical analysis

Statistical analysis was done using SPSS statistical package (SPSS, Chicago, IL). The frequencies of the alleles and genotypes among the case patients and control groups were determined and were compared by the chi-square test and Fischer's exact probability tests. ORs and their 95% confidence intervals (95% CIs) were calculated. ORs were estimated by the method of Haldane if required. Differences between groups for continuous variables were evaluated by Student's t-test.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Table 1 shows the clinical and demographic characteristics of 118 Italian patients with BD. We observed 37 (31.4%) patients with vascular lesions. The 2 most common lesions were subcutaneous thrombophlebitis (12/118, 10.2%) and DVT of the legs (25/118, 21.2%). Two patients had isolated intracardiac thrombosis and 1 patient had associated Budd-Chiari syndrome and extensive inferior vena cava and leg vein thromboses. Therefore, a total of 27 patients had evidence of DVT. Arterial lesions were not present. There were no significant differences between patients with and without DVT (Table 1).

Table 1. Demographic and clinical features of 118 Italian patients with Behçet's disease*
 Total BD (n = 118)BD without DVT (n = 91)BD with DVT (n = 27)
  • *

    Data presented as number (%) unless otherwise noted. BD = Behçet's disease; DVT = deep vein thrombosis.

  • Sixteen of 31 patients had isolated anterior uveitis.

  • Pathergy test was performed in 53 patients (35 without DVT and 18 with DVT).

  • §

    HLA typing was performed in 94 patients (74 without DVT and 20 with DVT)

Female/male54/64 (45.8./54.2)41/50 (45.1/54.9)13/14 (48.1/51.9)
Age at disease onset, mean ± SD, years30 ± 1229 ± 1131 ± 16
Disease duration, mean ± SD, years12 ± 812 ± 812 ± 10
Oral ulcer118 (100)91 (100)27 (100)
Genital ulcer72 (61.0)59 (64.8)13 (48.1)
Cutaneous lesions98 (83.1)75 (82.4)23 (85.2)
 Erythema nodosum49 (41.5)37 (40.7)12 (44.4)
 Papulopustular lesions67 (56.8)51 (56.0)16 (59.2)
Eye lesions74 (64.4)56 (61.5)20 (74.1)
 Anterior uveitis31 (26.3)26 (28.6)5 (18.5)
 Posterior uveitis/retinal vasculitis61 (51.7)44 (48.4)17 (63.0)
Arthritis52 (44.1)39 (42.9)13 (48.1)
Subcutaneous thrombophlebitis12 (10.2)10 (11.0)2 (7.4)
Central nervous system involvement24 (20.3)20 (22.0)4 (14.8)
Epididymitis6 (5.1)5 (5.5)1 (3.7)
Positive pathergy test result26 (49.0)17 (48.6)9 (50.0)
HLA–B51§54 (57.4)41 (55.4)13 (65.0)

The allele and genotype frequencies of prothrombin gene G20210A and factor V gene G1691A polymorphisms in BD patients and in the control group are shown in Table 2. The distributions did not differ significantly between BD patients and healthy controls.

Table 2. Allele and genotype frequencies of prothrombin gene G20210A and factor V gene G1691A polymorphisms in Italian Behçet's disease patients and controls*
VariableHealthy controls (n = 132)Behçet's disease (n = 118)POdds ratio (95% CI)
  • *

    Data presented as number with variable/total number possible (%). 95% CI = 95% confidence interval; NS = not significant.

  • Prothrombin gene G20210A polymorphism test was performed on 117 of 118 patients with Behçet's disease.

Factor V G1691A    
 Alleles    
  A5/264 (1.9)9/236 (3.8)NS1.4 (0.9–2.0)
  G259/264 (98.1)227/236 (96.2) 0.7 (0.3–1.4)
 Genotypes    
  AA0/1320/118  
  GA5/132 (3.8)9/118 (7.6)NS 
  GG127/132 (96.2)109/118 (92.4)  
Prothrombin G20210A    
 Alleles    
  A5/264 (1.9)8/234 (3.4)NS1.3 (0.8–2.0)
  G259/264 (98.1)226/234 (96.6) 0.7 (0.4–1.4)
 Genotypes    
  AA0/1320/117  
  GA5/132 (3.8)8/117 (6.8)NS 
  GG127/132 (96.2)109/117 (93.2)  

The comparisons of the frequencies of carriage rates of prothrombin gene G20210A and factor V gene G1691A polymorphisms in controls and in BD patients with and without DVT are shown in Table 3. No significant differences were found. No patient was carrying both factor V Leiden and prothrombin gene G20210A mutations.

Table 3. Comparisons of carriage rates of prothrombin gene G20210A and factor V gene G1691A polymorphisms in controls and Italian Behçet's disease patients with and without deep vein thrombosis*
Carriage rateBD with DVT (n = 27) ABD without DVT (n = 91) BControl (N = 132) CA versus C PB versus C PA versus B P
  • *

    Data presented as number (%). BD = Behçet's disease; DVT = deep vein thrombosis; NS = not significant.

  • Prothrombin gene G20210A polymorphism test was performed on 117 of 118 patients with Behçet's disease.

Factor V G1691A      
 A1 (3.7)8 (8.8)5 (3.8)NSNSNS
 G26 (96.3)83 (91.2)127 (96.2)   
Prothrombin G20210A      
 A2 (7.4)6 (6.7)5 (3.8)NSNSNS
 G25 (92.6)84 (93.3)127 (96.2)   

The HLA–B51 allele frequency was significantly higher in BD patients compared with healthy controls (57.4% versus 19.2%; P = 0.0001, OR 5.7, 95% CI 3.1–10.3). We also investigated possible associations of the 2 polymorphisms studied with BD with or without DVT stratifying on HLA–B51. Although this analysis was limited by the low number of patients studied, no significant associations were observed in HLA–B51-positive or HLA–B51-negative patients (data not shown).

The associations between prothrombin gene G20210A and factor V gene G1691A polymorphisms and BD clinical manifestations defined in Table 1 were evaluated in the 118 BD Italian patients comparing patients with and without manifestations. The frequency of 20210A allele was significantly higher in BD patients with ocular disease than in those without (10.8% versus 0%; P = 0.03, OR 11.4, 95% CI 5.2–24.9), particularly in the patients with posterior uveitis/retinal vasculitis (13.1% versus 0%; P = 0.004, OR 18.2, 95% CI 8.3–39.8).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

We observed a 31.4% prevalence of vascular lesions in our consecutive series of Italian patients with BD. This frequency is similar to that reported in other studies. In particular, Koç et al, in a study designed to evaluate the prevalence of vascular involvement in BD, showed a prevalence of 27.7% in a series of Turkish patients (4). The 2 most common lesions observed in our study were subcutaneous thrombophlebitis and DVT of the legs. Two patients had isolated intracardiac thrombosis and 1 patient had associated hepatic vein thrombosis and extensive inferior vena cava and leg vein thromboses. Similar to the experience cited by Bayraktar et al (26), this patient had a catastrophic course with death occurring a few months after the development of vascular lesions. Arterial lesions were not present in our series of Italian patients.

We also compared the demographic and clinical characteristics of the patients with and without DVT. No significant differences were found. Koç et al reported a statistically significantly higher frequency of pathergy positivity, erythema nodosum, and eye involvement in patients with vascular lesions (4). Eye lesions, in particular posterior uveitis/retinal vasculitis, were also more frequent in our Italian patients with DVT, although the difference was not significant.

The factor V Leiden mutation is the most common cause of inherited thrombophilia (12). Seven studies have evaluated factor V Leiden as a potential risk factor for DVT in BD patients (14–20) (Table 4). Three studies have examined Turkish patients (14–16), 1 study examined patients from Saudi Arabia (17), 1 studied Palestinian and Jordan patients (18), and 2 studies examined European patients (19, 20). The results were controversial. Two of the studies reported a significant association between factor V Leiden mutation and venous thrombosis in BD (14, 17), but in the 4 other studies this association was not confirmed (15, 18–20). The seventh study was limited by the low number of patients with DVT (only 5) (16). Two of the studies were well-planned, case-control studies that evaluated 2 series of Turkish patients with BD referred to Ankara and Istanbul Universities (14, 15). Their results were conflicting. Gül et al (14) showed that heterozigosity for factor V gene mutation was associated with an almost 6-fold increase in the risk of venous thrombosis, whereas Toydemfir et al (15) showed that this mutation was not a risk factor for venous thrombosis in BD.

Table 4. Prevalence of the prothrombin gene G20210A and factor V gene G1691A mutations in Behçet's disease patients with and without vascular lesions: results from the literature*
ReferenceGeographic originType of vascular lesionPrevalence in BD patients with VL APrevalence in BD patients without VL BPrevalence in controls CP (OR, 95% CI) A versus B
  • *

    Prevalence reported as number with variable/total number tested (%). BD = Behçet's disease; VL = vascular lesions; OR = odds ratio; 95% CI = 95% confidence interval; DVT = deep vein thrombosis; NS = not significant; NR = not reported; RVO = retinal vasoocclusion; AT = arterial thrombosis.

  • Patients without evidence of ocular disease.

  • The geographic origin of the patients is not clearly defined.

Factor V gene G1691A mutation      
 Gül et al (14)TurkeyDVT12/32 (37.5)3/32 (9.4)11/107 (10.3)0.008 (5.8, 1.4–23.2)
 Toydemir et al (15)TurkeyDVT10/30 (33.3)6/30 (20.0)4/100 (4.0)NS
 Öner et al (16)TurkeyDVT3/5 (60)7/39 (17.9)6/81 (7.1)NR
 Mammo et al (17)Saudi ArabiaDVT3/8 (37.5)0/15NR0.03 (NR)
 Verity et al (18)Palestine, JordanDVT8/24 (33.3)21/82 (25.6)23/120 (19.2)NS
  RVO11/25 (44.0)5/31 (16.1) 0.02 (1.06, NR)
 Lesprit et al (19)FranceDVT, AT0/15---
 Espinosa et al (20)SpainDVT, stroke0/140/242/100 (2.0)NS
 Present studyItalyDVT1/27 (3.7)8/91 (8.8)5/132 (3.8)NS
Prothrombin gene G20210A mutation      
 Gül et al (24)TurkeyDVT10/32 (31.3)1/32 (3.1)-0.003 (14.1,1.7–118.2)
 Toydemir et al (15)TurkeyDVT1/30 (3.3)0/303/100 (3.0)NS
 Espinosa et al (20)SpainDVT, stroke1/14 (7.1)0/241/100 (1.0)NS
 Present studyItalyDVT2/27 (7.4)6/90 (6.7)5/132 (3.8)NS

In our study, we did not find any association between factor V Leiden mutation and venous thrombosis in Italian patients with BD. Similarly, the factor V gene mutation was not found in 2 series of BD patients with thrombosis, 1 from France (15 patients), the other from Spain (14 patients) (19, 20). Therefore, the factor V Leiden mutation seems not to be a risk factor for venous thrombosis in European patients with BD.

Toydemfir et al (15) found a high frequency of factor V Leiden mutation in BD patients without DVT and suggested an association between this mutation and BD. In our study, the frequencies of factor V Leiden mutation in the total BD patients and in the subgroup without DVT were similar to that of controls.

Another study established inherited cause of thrombophilia is the G20210A mutation in the 3′-untranslated region of the prothrombin gene (21). We described 2 cases of BD with thrombotic lesions that were heterozygous for the prothrombin G20210A mutation (27). We thus decided to plan the present study to confirm a possible association. We did not find any significant differences in the frequency of prothrombin gene mutation between Italian BD patients with and without DVT. A recent study from Spain, confirming our results, did not find any association between this mutation and thrombosis (prevalently DVT) in patients with BD (Table 4) (20). Discordant data are reported on Turkish BD patients. A heterozygous prothrombin gene G20210A mutation was found to be significantly associated with DVT by Gül et al (24); this was not the case in the Toydemfir et al study (15). In these 2 Turkish studies, there were large differences in the prevalence of this mutation in BD patients with and without DVT (31.3% versus 3.3%).

There are different possible explanations for the conflicting results regarding the association of these 2 thrombophilic mutations with BD; allelic heterogeneity existing between ethnic groups, modification of the association by other genetic or environmental factors that vary between the populations studied, selection bias in sampling of cases or controls, and publication bias. However, one of the most important causes of failure to replicate findings in genetic association studies is the inadequacy of sample sizes (28). So far, the maximum number of patients with DVT studied has been only 32. To test the association between thrombophilic gene polymorphisms and DVT in BD, a larger group of cases with DVT would be needed. Therefore, multicenter collaborations to recruit an adequate number of cases are required.

In our study, we also evaluated whether prothrombin gene G20210A and factor V gene G1691A polymorphisms were associated with specific clinical findings comparing patients with and without certain manifestations. The frequency of the 20210A allele was significantly higher in BD patients with ocular disease than in those without. These data suggest that a prothrombin gene G20210A mutation is a risk factor for the development of ocular disease. However, because the strength of the association was higher in BD patients with more severe ocular disease (posterior uveitis/retinal vasculitis), this polymorphism may also influence the severity of ocular involvement. Additional studies are needed to replicate the association between gene G20210A mutation and ocular disease. Recurrent vascular occlusion due to an obliterative retinal vasculitis and vascular thrombosis is a major cause of visual loss in BD. A prothrombotic condition may be implicated in the pathogenesis of ocular inflammatory disease in BD.

Verity et al (18) found a significant association between factor V Leiden mutation and retinal vascular occlusion in BD (Table 4). However, the strength of the association was weak (OR 1.06) and the significance was lost upon comparison of ocular patients with and without vasoocclusive disease. We failed to find any association between this polymorphism and ocular lesions.

In conclusion, our study shows a frequency of vascular lesions in Italian BD patients similar to that reported in studies from other countries. However, we did not find any association between venous thrombosis and the factor V Leiden mutation or the G20210A mutation in the 3′-untranslated region of the prothrombin gene. Thrombotic lesions in Italian BD patients seem more probably related to the endothelial activation induced by the vasculitic process. Furthermore, our data support the hypothesis that prothrombin gene G20210A mutation may influence the development and severity of ocular involvement in BD.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES
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