Vascular Behcet and Mutations in Thrombogenic Genes: Methylene Tetrahydrofolate Reductase,Factor V,and Prothrombin

Abstract

Vasculitis, thrombophlebitis, arterial aneurysms, and occlusions occur in about 25% of patients with Behçet's disease (BD). The common inherited gene defects, factor V (FV) 1691A (Leiden), methylene tetrahydrofolate reductase (MTHFR) 677T, and prothrombin 20210A, are known risk factors for thrombosis. The aim of the study was to evaluate the contribution of these mutations to thrombosis in Israeli patients with BD. Fifty-four patients with BD (n=54; 27 men and 27 women) underwent clinical and genetic evaluation. Most patients (n=43; 79.6%) were of Arab descent (31 sporadic and 12 familial cases from 4 families), and 11 patients (20.4%) were of Jewish descent (all sporadic cases). The FV Leiden mutation was identified in five patients (9.2%), and eight patients were MTHFR 677TT homozygotes (14.8%). None had the 20210A mutant prothrombin allele. No statistical differences between carriers and noncarriers with regards to demographic and disease manifestations were calculated. Arabs were diagnosed earlier than Jewish patients (25.8±11.6 compared with 37.2±10.7, p=0.01, respectively), but Jewish patients had, respectively, more events of deep vein thrombosis (DVT) compared with Arabs (3 of 11, 27.3% and 3 of 43, 7%, p=0.09). Thrombotic events in our patients with BD were not associated with variations in thrombophilic genes.

Introduction

Behcet's Disease (BD) is a multisystemic, relapsing, inflammatory vasculitic disorder traditionally described as consisting of recurrent oral and genital ulcerations, folliculitis, erythema nodosum, and uveitis. Vasculitis occurs in about 25% of patients (Ames et al., 2001; Mendoza-Pinto et al., 2010). The vasculopathy of BD is unique, and any type of vessel can be involved (Koç et al., 1992; Ames et al., 2001; Espinosa et al., 2002; Kapsimali et al., 2010). The vascular lesions bear an occlusive nature, but the mechanism of hypercoagulability remains basically unknown (O'Duffy, 1994; Kaklamani et al., 1998; Koné-Paut et al., 2007; Yazici et al., 2007; Mendoza-Pinto et al., 2010). Given that, an association between the occurrence of thrombosis and a primary abnormality in the coagulation pathway was inconsistently described in patients with BD. Levels of protein C and protein S antithrombin activity, usually normal in patients with BD (Guermazi et al., 1998; Nalçaci and Pekçelen, 1998; Mader et al., 1999; Houman et al., 2001; Leiba et al., 2004; Ahn et al., 2008), were found to be reduced in patients with BD with venous thromboembolism compared with patients with BD without thrombosis (Chafa et al., 1992; Navarro et al., 2004; Caramaschi et al., 2010). Protein C deficiency was reported in BD cases complicated by extensive venous thrombosis (Shehto et al., 1992) and mesenteric thrombosis (Bayraktar et al., 1998).

Several genetic defects have been associated with an increased risk for venous thrombosis and arteriosclerosis (Bertina et al., 1992, 1994; Zoller et al., 1994; Frosst et al., 1995; Poort et al., 1996; Bertina, 1997; Uthman et al., 2006). Common inherited risk factors for thrombosis include factor V Leiden (FVL), factor II prothrombin gene G20210A polymorphism, and the C677T variant of methylene tetrahydrofolate reductase (MTHFR) (Bertina et al., 1992, 1994; Zoller et al., 1994; Frosst et al., 1995; Poort et al., 1996; Bertina, 1997). FVL, a nucleotide replacement in position 1691, resulting in the substitution of arginine (R) for glutamine (Q) at position 506, conveys resistance to the action of activated protein C, and, thus, leads to an increased risk for thrombosis (Zoller et al., 1994). The prothrombin gene encodes the serine protease thrombin, a key enzyme in the processes of hemostasis and thrombosis. Prothrombin gene G20210A polymorphism is associated with a high risk for venous thrombosis (Bertina et al., 1994; Poort et al., 1996). The MTHFR catalyzes the reduction of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the cofactor for the methylation of homocysteine to methionine. Individuals homozygous for the thermolabile variant of MTHFR, which results from a common mutation [Ala677→Val (C677T)] and is found in 5%-15% of the general population, have significantly elevated plasma homocysteine levels and are at a higher risk for the development of arterial disease and also for venous thrombosis (Frosst et al., 1995). Higher levels of homocysteine were described in several cohorts of vascular Behcet (La Regina et al., 2010).

The presence of an inherited coagulation defect that may increase the risk of thrombotic events in BD was occasionally described (Leiba et al., 2004; Ozkul et al., 2005; Espinosa et al., 2010). Genetic evaluation of a 39-year-old woman with BD, presenting with deep vein thrombosis of the leg, revealed homozygosity for the G20210A polymorphisms in the prothrombin gene (Uthman et al., 2006). This mutation was found to be over-represented in patients with BD and thrombosis (Ricart et al., 2006). Several studies either supported or refuted an association between FVL, prothrombin G20210A mutation, and homozygosity for 677T allele with thrombosis, in patients with BD (Espinosa et al., 2002; Ateş et al., 2003; Leiba et al., 2004; Ricart et al., 2006; La Regina et al., 2010).

In order to assess the contribution of inherited thrombophilic risk factors to thrombosis in BD, we studied the frequencies and distribution of MTHFR C677T, FVL G1691A, and prothrombin G20210A polymorphisms in Israeli patients with BD who were of Jewish and Arab origin.

Patients and Methods

Patients

Patients with BD were ascertained by using the criteria proposed by the International Study Group for Behcet's Disease (ISBD, 1990) that include oral aphthae (at least three episodes in 1 year) plus at least two of the following: genital ulceration, erythema nodosum, necrotic folliculitis, uveitis, and retinal vasculitis. Patients were recruited at the Department of Rheumatology, Bnei-Zion Medical Center Haifa, and the Institute of Human Genetics, RAMBAM Health Care Campus, during 2006-2008. Data related to the entire spectrum of disease manifestations were collected from medical files and patient interviews. Patients were given educational information about the aims of the study, signed an informed consent form, and a blood sample was drawn for molecular testing.

Genetic testing

Polymerase chain reaction and restriction fragment length polymorphisms for the predominant mutations in MTHFR, FVL, and the prothrombin genes were performed.

MTHFR mutation

The C677T predominant mutation in the MTHFR gene was determined as described earlier (Frosst et al., 1995). The C to T substitution creates a Hinf1 restriction site. Primers 5′-TGAAGGAGAAGGTGTCTGCGGGA-3′ and 5′-AGGACGGTGCGGTGAGAGTG-3′ were used to generate a 198bp product. The mutant allele digested by Hinf1 yields two fragments of 175 and 23 bp.

FVL mutation

The G1691A predominant mutation in factor V was performed as described earlier (Zoller et al., 1994), by using forward 5′-CATACTACAGTGACGTGGAC-3′, and reverse 5′-TGTTCTCTTGAAGGAAATGC-3′. The 206 bp amplified product was subjected to MnlI digestion, which in a normal allele produced fragments of 122, 47, and 37 bp. A G to A mutation (nucleotide position 1691) in the codon for Arg506 resulted in the loss of one cleavage site, generating fragments of 159 and 47 bp (pattern in a homozygous individual). Analysis of an heterozygous allele yielded bands of 159, 122, 47, and 37 bp.

Prothrombin factor II mutation

The G20210A predominant mutation in prothrombin factor II (Poort et al., 1996) was studied by using forward 5′-TGTGTTCCGCCTGAAGAAGTG-3′ and reverse mismatched 5′-ATAGCACTGGGAGCATTGAa*GC-3′ (the A replaces a G to create a HindIII restriction site for the mutant allele). A product of 175 bp is restricted by HindIII yielding two fragments of 152 and 23 bp.

Data management and statistical analysis

The BD severity score was calculated according to Krause et al. (2001) as mild, moderate; and severe symptoms were awarded one, two, or three points, respectively. Pearson correlation coefficient (r) and its significance (p) were calculated between age at diagnosis and severity score for each patient. Chi-square test or Fisher's exact test was performed, when appropriate, to analyze a statistically significant relationship between demographic (e.g., origin, gender), genetic (carriage of FVL, prothrobin factor II, and MTHFR), and categorical clinical variables (e.g., deep vein thrombosis [DVT], superficial thrombosis, arthritis, etc). A t-test was done to test statistically significant differences in mean continuous variables (e.g., age at disease onset, disease duration, or severity score) in carriers compared with noncarriers. p-values<0.05 were considered statistically significant.

Results

Table 1 describes the demographic and clinical manifestations of 54 patients, all of whom fulfilled ISBD criteria for BD. The cohort consisted of 27 (50%) men and 27 (50%) women. Most patients (n=43; 79.6%) were of Arab descent (31 sporadic and 12 familial cases from 4 families), and 11 patients (20.4%) were of Jewish descent (all sporadic cases). Mean age at diagnosis was 27.9±12.1 years. Other than ulcers (oral and genital), the most common clinical manifestations, in descending order, were 38 arthralgia (70.4%); 32 skin lesions (erythema nodosum, folliculitis, papulopustular lesions, or others) (59.3%); 27 arthritis (50.0); and 22 ocular disease (anterior uveitis or pan uveitis) (40.8%). Vascular lesions were reported by 14 patients (25.9%). Of these, five (9.2%) had DVT, four (7.5%) had vasculitis, three (5.6%) had major vein thrombosis events, one (1.85%) patient had superficial vein thrombosis and one patient had both DVT and superficial vein thrombosis (1.85%). None had arterial thrombosis. The clinical characteristics were similarly manifested in patients with BD with or without vascular events (Table 1).

Table 1.

Demographic and Clinical Characteristics in 54 Patients with Behçet's Disease Related Thrombosis

	BD patients with thrombosis n=11 (20.4%)	BD patients without thrombosis n=43 (79.6%)	p-Value
Demographic
Male/female ratio (27/27, 1.0)	8/3 (2.7)	19/24 (0.79)	0.175
Ethnic descent (n,%)
Jews (11, 20.4%)	4 (36.4)	7 (16.3)	0.338
Ashkenazi (4, 7.4%)	1 (9.1)	3 (7.0)
Non-Ashkenazi (7, 13.0%)	3 (27.3)	4 (9.3)
Arabs (43, 79.6%)	7 (63.6)	36 (83.7)
Muslims (34, 63.0%)	7 (63.6)	27 (62.8)
Druze (6, 11.1%)	—	6 (14.0)
Christians (3, 5.5%)	—	3 (7.0)
Clinical manifestation according to ISBD
Age at disease onset (years)
Range (1-50)	15-40	1-50
Median (28.0)	29.5	27.0
Mean±SD (27.9±12.18)	29.6±7.9	27.6±13.0	0.638
Years since initial diagnosis
Range (1-35)	4-13	1-35
Median (10.0)	8.0	10.0
Mean±SD (11.18±8.0)	8.5±3.3	11.7±8.8	0.064
Oral aphtosis (54, 100.0%)	11 (100)	43 (100)	0.204
Genital ulcer (44, 81.5%)	10 (90.9)	34 (79.1)	0.367
Skin lesion (32, 59.3%)	8 (72.7)	24 (75.0)	0.308
Arthralgia (38, 70.4%)	7 (63.6)	31 (72.1)	0.584
Arthritis (27, 50.0%)	7 (63.6)	20 (46.5)	0.501
Anterior Uveitis (17, 31.5%)	4 (36.4)	13 (25.6)	0.491
Panuveitis (5, 9.3%)	1 (9.1)	4 (9.3)	0.573
Recurrent headaches (7, 13.0%)	1 (9.1)	6 (14.0)	0.668
Gastrointestinal bleeding (None)	—	—
Neuro Behcet (None)	—	—
Bowel perforation (None)	—	—

BD, Behçet's disease; ISBD, International Study Group for Behcet's Disease; SD, standard deviation.

The FVL was identified in five patients (9.2%), all of Arab origin. The 677T mutant allele in MTHFR was identified in 26 (49%) patients including heterozygotes (n=18, 34%) and homozygotes (n=8, 15%). None had the factor II prothrombin 20210A mutant allele. No statistically significant differences between carriers and noncarriers with regard to gender, origin, age at diagnosis, disease manifestations, and severity score were calculated (Table 2).

Table 2.

Thrombotic Events in the 54 Patients with Behçet's Disease Associated to Predominant Mutations in Factor V Leiden and Methylene Tetrahydrofolate Reductase

	FVL Arg506Gln mutation
Variable	Carriers n=5 (9.2%)	Noncarriers n=49 (90.8%)	Significant test
Age at diagnosis^a	21.4±8.7	29.5±12.2	p=0.3
Gender
Female	2 (7.4)	25 (92.6)
Male	3 (11.1)	24 (88.9)	p=0.48
Origin
Arabs	5 (11.6)	38 (88.4)
Jews	0	11 (100)	p=0.26
DVT
Yes	0	6 (100)
No	5 (10.4)	43 (89.6)	p=0.38
Major vein Thrombosis
Yes	0	3 (100)
No	5 (9.8)	46 (90.2)	p=0.72
Superficial vein thrombosis
Yes	1 (50)	1 (50)
No	4 (7.7)	48 (92.3)	p=0.2
Vasculitis
Yes	1 (25)	3 (75)
No	4 (8.0)	46 (92)	p=0.31

	MTHFR C677T mutation
	Carriers (n=26)		Noncarriers (n=28)
	677TT n=8 (15%)	C677T n=18 (34%)	CC677 n=28 (51%)
Age at diagnosis^a	26.4±9.7	26.3±12.9	29.5±12.5	p=0.64
Gender
Female	6 (22.2)	11 (40.7)	10 (37)
Male	2 (7.4)	8 (29.6)	17 (63)	p=0.1
Origin
Arabs	6 (14)	15 (34.8)	22 (51.2)
Jews	2 (18.2)	4 (36.4)	5 (45.5)	p=0.48
DVT
Yes	1 (16.7)	3 (50)	2 (33.3)
No	7 (15)	15 (32)	26 (53)	p=0.6
Major vein thrombosis
Yes	0	0	3 (100)
No	8 (16)	18 (36)	26 (48)	p=0.2
Superficial vein thrombosis
Yes	0	1 (50)	1 (50)
No	8 (15.7)	17 (33.3)	27 (51)	p=0.8
Vasculitis
Yes	1 (50)	1 (25)	2 (50)
No	7 (14.3)	17 (34.7)	26 (48)	p=0.8

Ages at diagnosis describe as mean±SD.

FVL, factor V Leiden; MTHFR, methylene tetrahydrofolate reductase; DVT, deep vein thrombosis.

Differences in disease manifestations were found among Arab and Jewish patients with BD. These include earlier age at onset in Arab patients (25.8±11.6) compared with Jewish (37.2±10.7) patients (p=0.01). The DVT was observed more frequently in Jewish patients compared with Arab patients (3 of 11, 27.3% and 3 of 43, 7%, p=0.09, respectively) as was arthritis (8 of 11, 72.7% compared with 19 of 43, 44.2%, p=0.088, respectively). Thrombotic events other than DVT were equally manifested by Jewish and Arab patients with BD (Table 3).

Table 3.

Clinical Characteristics of Arabs Compared with Jewish Patients with Behçet's Disease

Variable	Arabs n=43 (89.6%)	Jews n=11 (20.4%)	Significant test
Age at diagnosis^a	25.8±11.6	37.2±10.7	p=0.01
Gender
Female	22 (51.2)	5 (45)
Male	21 (48.8)	6 (55)	p=0.73
DVT
Yes	3 (7)	3 (27.3)
No	40 (93)	8 (72.7)	p=0.09
Major vein thrombosis
Yes	2 (4.6)	1 (9)
No	41 (95.4)	10 (91)	p=0.5
Superficial vein thrombosis
Yes	2 (4.6)	0
No	41 (95.4)	11 (100)	p=0.3
Vasculitis
Yes	3 (7)	1 (9)
No	40 (93)	10 (91)	p=0.61
Arthralgia
Yes	30 (70)	8 (72.7)
No	13 (30)	3 (27.3)	p=0.58
Arthriris
Yes	19 (44.2)	8 (72.7)
No	24 (55.8)	3 (27.3)	p=0.088

Ages at diagnosis describe as mean±SD.

Discussion

Although the mechanisms of either venous or arterial thrombosis, in either small or large vessels, seem to be heralded by vaculitis (Ehrlich, 1997) the etiopathology of vascular thrombosis in BD is basically unclear. The efforts to locate genetic factors associated with vascular Behcet remain challenging. So far, significant associations between FVL and prothrombin 20210A mutant allele with vascular Behcet were reported from Turkey, where both the disease itself and FVL are prevalent (Kiraz et al., 2002). Conversely, no association between vascular Behcet and known polymorphisms in thrombophilia genes was reported in BD cohorts from Sheba Tel Hashomer in Israel (Leiba et al., 2004) and others from Italy, Spain, and likewise from Turkey (Oner et al., 1998; Toydemir et al., 2000; Espinosa et al., 2002; Silingardi et al., 2004). In this study, none of the predominant mutations in MTHFR, factor V, and prothrombin genes were found to be associated with thrombotic events of our patients with BD. Interestingly, our cohort was mainly composed of Arab patients (n=43; 79.6%), of whom 5 patients had FVL (11.6%) and none carried 20210A, a proportion in line with the expectation based on prevalence studies in the general Arab population (Dagan et al., 2006). We, thus, conclude that the overall high carrier frequency, for FVL, in our cohort of patients in whom BD was diagnosed, should be attributed to their Mediterranean extraction rather than related to BD (Touitou et al., 2000; Atagunduz et al., 2003; Imirzalioglu et al., 2005; Rabinovich et al., 2007; Ayesh et al., 2008). Homozygosity for the 677T mutant allele in MTHFR and the consequent increased level of homocysteine is considered a risk factor for vascular and arterial occlusion, at large (Bertina et al., 1992; Mendoza-Pinto et al., 2010), and in patients with BD. Here, 15% of patients with BD were 677T homozygotes (8 of 54 patients with BD) compared with 6.7% in the general population (7 of 104) p>0.05 (derived from our historical control group). However, when Arab patients with BD were separately analyzed (n=43), six 677T homozygotes (13.9%) were identified as compared with (2 of 60; 3.3%) in our historical control group, p=0.05. Despite the significantly higher prevalence of this mutation in Arab patients with BD compared with controls, no association with thrombosis was calculated. This finding supports other observations (e.g., Toydemir et al., 2000; Koné-Paut et al., 2007) and should be further investigated with regard to homocysteine levels.

The clinical characteristics noted in Arab and Jewish patients with BD differed in some aspects. Earlier age at diagnosis was reported in Arabs (25.8±11.6) compared with Jews (37.2±10.7), p=0.01. Jewish patients, although rare here (n=11; non-Ashkenazi Jews n=8; Ashkenazi Jews n=3), had higher rates of articular and deep vein thrombosis events compared with Arab patients with BD. These differences did not reach significance; however, it may indicate a trend. Our results are in accord with those of Krauze et al. (2001), who have previously commented on the relative expression of BD among the different ethnic groups in Israel and have shown that Jewish patients with BD from North African countries manifested a more severe disease compared with Ashkenazi patients, especially with regard to the frequency of ocular disease, arthritis, neuro-Behcet, and deep vein thrombosis.

Our results corroborate most studies from Mediterranean countries and demonstrate low frequencies of FVL and prothrombin 20210A carriage and no association with thrombotic events in patients with BD. The highly significant prevalence of MTHFR 677T homozygosity in our Arab population is not associated with thrombophilic events. Our small number of patients warrants further investigation.

Footnotes

Disclosure Statement

No competing financial interests exist.

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