The relation of PON 1L 55 M gene polymorphism and clinical manifestation of Behcet ’ s disease

Purpose: Behçet’s disease is a multisystem disease characterized by recurrent oral and genital ulcers, relapsing uveitis, mucocutaneous, articular, gastrointestinal, neurologic, and vascular manifestations. Paraoxonase is believed to play an important role in protection of LDL and HDL particles from oxidation, in antioxidant effect against lipid peroxidation on cellular membranes, and in anti-inflammatory process. Lipid peroxidation and free oxygen radicals have been thought to play a role in pathogenesis of BD. The association of paraoxonase gene polymorphisms with Behçet’s Disease in a group of Turkish patients with clinical manifestations and healthy controls has been investigated. Patients and Methods: Paraoxonase (PON-1-L55M) gene polymorphism was investigated in 50 Behcet patients and 50 healthy individuals with a PCR/RFLP method. Results: There were significant differences between patients and the control group in allele frequencies of the PON1 L55M polymorphism (p=0.04). Also, when patients were compared with the control group according to clinical manifestations, this statistical significance was getting sharper. Compared with the PON55 L allele, the M allele was associated with greater than 3.5 fold (OR 3.5, 95% CI 1.3–8.9) increased risk of ocular (OR 2.4, 95% CI 1.1–5.3), 2.4 fold joint and 3.1 fold (OR 3.1, 95% CI 1.1–8.4) central nervous system manifestations of BD. Conclusion The PON L55M gene polymorphism seemed to play a role in the pathogenesis of BD.


INTRODUCTION
Behçet's disease (BD) is described as a chronic unclassified systemic vasculitis.Although it was originally established with recurrent oral and genital ulcers and uveitis, it is now accepted as a multisystem disorder also affecting all types and sizes of blood vessels, joints, lungs, central nervous system, and intestines (Sakane et al., 1999).In about 20-35% of cases of BD, diverse vascular complications, such as a deep vein thrombosis, myocardial infarction, arterial aneurysm, and arterial thrombus formation have been diagnosed (Kural-Seyahi et al., 2003).Vascular and cardiac events are partly due to atherosclerosis (Oztürk et al., 2006).In patients with BD, the mechanism of atherosclerosis may be attributed to lipid abnormalities.Lipids and lipoprotein peroxidation are considered to be important in the pathogenesis of atherosclerosis (Loeper et al., 1983).Their profiles and relation with atherogenesis was described in patients with BD (Orem et al., 1995;Mitamura et al., 1988).
Paraoxonase 1 (PON1) is an enzyme exclusively located on high-density lipoprotein (HDL) in the serum (Ruiz et al., 1995).PON1 hydrolyzes organophosphate substrates and metabolizes lipid peroxides leading to protection against accumulation of low-density lipoprotein (LDL) that otherwise might lead to atherosclerotic plaque formation (Mackness et al., 1997).Major polymorphisms of PON1 include the replacement of Gln (Q) by Arg (R) at position 192, and that of Leu (L) by Met (M) at position 55.For the L55M PON1 polymorphism, the L allele carriers were found to have higher mRNA levels (Adkins et al., 1993;Humbert et al., 1993) and accordingly, the L allele carriers have significantly higher enzyme concentrations (Eckerson et al., 1983).When compared to PON55M isoform, PON55L is associated with higher serum activity, higher stability and resistance to proteolysis.Furthermore PON55L plays an important role in the packing of the protein correctly (Harel et al., 2007).A relationship between PON1 genotypes and the antioxidant activity of HDL has also been demonstrated (Kuremoto et al., 2003).
Functional polymorphisms in the PON1 gene are attractive candidates due to their impact on antioxidant activity of HDL and subsequently on inter-individual vascular disease susceptibility.Common polymorphisms in paraoxonase 1 gene are described as risk factors in a variety of vascular disorders including coronary artery disease and carotid artery stenosis.However, to our knowledge, no investigation has been undertaken on the association between the PON1 L55M single nucleotide polymorphism and BD.In this study, the possible associations between PON1 L55M polymorphism and Turkish BD patients and its clinical manifestations have been investigated.

MATERIALS AND METHOD
Fifty patients with Behçet's disease and fifty healthy, unrelated control subjects who were all Turkish were included in this study.Patients with Behçet's disease were all fulfilling three or more of the International Study Group criteria for BD and were clinically diagnosed by Department of Dermatology.Clinical characteristics of both populations are shown in Table 1.
Genomic DNA was extracted from peripheral blood leucocytes (200 µl of total blood) by using Macherey-Nagel Nucleospin blood ® DNA extraction kit (Cat no.740.951.250)according to manufacturer's instructions.The DNA purity and concentration were determined by spectrophotometric measurement of absorbance at 260 and 280 nm.The PCR product was amplified with primers PON1-F 5′-CCT GCA ATA ATA TGA AAC AAC CTG 3′ and PON1-R 5′ TGA AAG ACT TAA ACT GCC AGTC-3′.Amplifications were performed in 0.2 ml thin-wall tubes of 50 µl aliquots containing 50 mM KCl, 10 mM Tris/HCI, 1.5 mM MgCl 2 , 0.5 µM of each of the four deoxynucleotides, 50 pmol of each primer (PON1-F and PON1-R), 1 U of Taq DNA polymerase and 20 ng genomic DNA.After an initial 4 min denaturation step at 94°C, 32 PCR cycles were run, each consisting of: 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min.A 72°C elongation was performed for 4 min at end of the PCR cycles.The PCR products of subjects and positive/negative controls were checked on a 1.5% agarose gel for the assay completion and then the PCR products of 172 bp were digested with restriction enzyme NlaIII by overnight incubation at 37°C.
NlaIII recognizes the CATG sequence corresponding to the Met allele.The Leu/Leu homozygote was identified by the presence of an uncut 172 bp band, whereas the Leu/Met heterozygote produces all three bands (172, 106, and 66 bp) following restriction digestion.The digestion products were electrophoresed on 3.5% agarose gel and visualized by staining with ethidium bromide and evaluated using a gel documentation system (Syngene, Genegenius Bio Imaging System).
Statistical analysis.A case-control study was performed and allelic frequency of the polymorphism was calculated both in case and control samples.The χ 2 test was used to compare allele frequency of the PON55 gene polymorphism between BD patients and controls.95% confidence interval (CI) was calculated to compare BD risk around genotypes and alleles.P value less than 0.05 was considered as statistically significant.The software used for the calculations was SPSS version 18 (SPSS Inc., Chicago, IL).

RESULTS
Age and gender matched 50 subjects with Behçet's disease (26 women and 24 men), and 50 healthy control subjects (27 women and 23 men) were genotyped for the PON55 (rs854560) SNP.The distribution of the genotypes in the controls was in Hardy-Weinberg equilibrium.The mean age (± S.D.) was 38.5±10.16 in patients, and 36.3±10.03 in control subjects (Table 1).
The frequencies of L and M alleles were 75.0% and 25.0% in cases, and 87.0 % and 13.0% in controls, respectively (Table 2) and the difference in allele frequency was significant (p=0.04).The relative risk for BD patients was more than 2.23 times higher (OR 2.23, 95% CI 1.07-4.66) in individuals with the PON55 M allele compared to the L allele.
Also, patients were compared with the control group according to clinical manifestations (Table 3  and 4).Frequency of the L allele was 65.6%, 73.5%, 67.9% and of the M allele was 34.4%, 26.5%, 32.1% in ocular, joint and central nervous system manifestations of BD patients, respectively.In the ocular, joint and central nervous system manifestations of BD patients, the frequency of the PON55 M allele was higher in comparison with that of the control group and the difference was significant (Table 3,p=0.01,p=0.04,p=0.02,respectively).Compared with the PON55 L allele, the M allele was associated with greater than 3.5 fold (OR 3.5, 95% CI 1.3-8.9)increased relative risk of ocular, 2.4 fold (OR 2.4, 95% CI 1.1-5.3)joint and 3.1 fold (OR 3.1, 95% CI 1.1-8.4)central nervous system manifestations of BD.The statistical significance was not present when other clinical manifestations of Behçet patients were compared with the control group (p>0.05).

DISCUSSION
The etiology and pathogenesis of Behçet's disease (BD) are not yet well understood.Histopathologic studies have established that vasculitis is the predominant lesion, affecting both the vessel wall and perivascular tissues (Sakane et al., 1999).Growing evidence indicates that oxidative stress is increased in BD, relating to overproduction of reactive oxygen products (ROS) and decreased efficiency of antioxidant resistance (Kose et al., 1951;Orem et al., 1997;Niwa et al., 1982).It has been demonstrated in in vitro studies that activated leucocytes form a large number of free oxygen radicals and this in turn causes endothelial cell damage.ROS can attack and damage a variety of critical biological molecules, includ-ing lipids, essential cellular proteins and DNA.Under oxidative stress, LDL and other serum lipoproteins, including HDL, are prone to lipid peroxidation.Recent studies show that lipid peroxidation in the serum of patients with BD was increased (Kose et al., 1951;Orem et al., 1997;Kose et al., 2001).
PON-1 is a serum enzyme bound with high density lipoproteins (HDL) and has been closely linked to the control of oxidative stress and inflammation, mainly at the circulation level (NgD et al., 2008).PON 1 protects lipoproteins against oxidative stress and makes possible to metabolize lipid peroxides that are largely distributed among tissues such as the liver, kidney, and intestine; but it is also present in plasma.There is a 10-to 40-fold inter-individual variability in serum PON1 activity (Hum- bert et al., 1993).PON1 gene polymorphism is one of the sources of this variability.When compared to PON1-55M allele, PON1-55L is correlated with higher PON1 activity and mRNA levels (Leviev et al., 1997;Li et al., 2000;Leviev et al., 2001).Karakucuk and coworkers (2004) and Mungan (2006) and coworkers found a decreased serum PON1 activity in BD patients in comparison with healthy controls.Decreased PON1 could explain the increased lipid peroxidation and oxidative stress observed in BD.This suggests a pathogenic mechanism that is supported by our study, where we show a significant correlation between the PON1 gene polymorphism and BD patients.Our study shows that carriers of the PON1 M allele have a 2.23 fold increased relative risk for developing BD.To our knowledge, this is the first study that investigates the possible associations between PON 55 polymorphism with Behçet's patients in a Turkish population.
PON1 is an HDL-associated enzyme which is able to hydrolyze organophosphates.Due to its functions in protecting LDL against oxidation, PON1 is also an antioxidant.Decrease in the levels of paraoxonase enzyme is a great risk for patients with cardiovascular diseases, rheumatoid arthritis, gout, and age-related macular degeneration (Ekinci et al., 2009;Jiang et al., 2011).Karakucuk et al. also found a decreased serum PON1 activity in BD patients with ocular involvement in comparison with healthy controls.PON is also important in metabolism as an organophosphate hydrolyser.Thus, PON1 protects the nervous system against organophosphate toxicity.Therefore, when patients with ocular, joint and central nervous system involvement were compared to the control group, this statistical significance was getting sharper.
Although the result of this study is statistically significant, because of the rarity of the Behçet's disease the sample size is considered as a limitation.In addition, further studies with different ethnic populations should be performed to validate whether there is a relationship between Behcet's disease and PON1 L55M gene polymorphisms.
Our findings suggest that PON1 L55M polymorphism is associated with an increased relative risk for BD.This finding was getting sharper when ocular, joint and central nervous system involvement was considered separately.Polymorphism in the PON1 gene might contribute to the reduced PON activity that causes increased lipid peroxidation and oxidative stress and inflammatory endothelial changes observed in patients with BD.