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Jul 2, 2010 - Etanercept reduces matrix metalloproteinase-9 level in children with polyarticular juvenile idiopathic arthritis and TNF-α-308GG genotype.

J Physiol Biochem (2010) 66:173–180 DOI 10.1007/s13105-010-0022-x


Etanercept reduces matrix metalloproteinase-9 level in children with polyarticular juvenile idiopathic arthritis and TNF-α-308GG genotype Jelena Basic & Dusica Pavlovic & Tatjana Jevtovic-Stoimenov & Jelena Vojinovic & Gordana Susic & Ivana Stojanovic & Gordana Kocic & Vuk Milosevic & Tatjana Cvetkovic & Milena Marinkovic & Andrej Veljkovic

Received: 15 December 2009 / Accepted: 11 May 2010 / Published online: 2 July 2010 # University of Navarra 2010

Abstract Genetic contribution of tumor necrosis factor polymorphism (TNF-α-308G/A) in patients with juvenile idiopathic arthritis (JIA) on response to TNF blocking agents, as well as matrix metalloproteinase-9 (MMP-9) production, is not yet well established. We have investigated whether the TNF-α-308G/A polymorphism can influence MMP-9 level and clinical response to etanercept (TNF receptor II-Fc fusion protein) in JIA patients, after 1 year of treatment. A total of 66 patients with polyarticular JIA and 65 healthy children were screened for the polymorphism using the polymerase chain reaction–restriction fragment length polymorphism method. JIA patients donated paired J. Basic (*) : D. Pavlovic : T. Jevtovic-Stoimenov : I. Stojanovic : G. Kocic : T. Cvetkovic : M. Marinkovic : A. Veljkovic Institute of Biochemistry, Medical Faculty, University of Nis, Bulevar dr Zorana Djindjica 81, 18000 Nis, Serbia e-mail: [email protected] J. Vojinovic Clinic of Pediatrics, Clinical Centre Nis, Nis, Serbia G. Susic Institute of Rheumatology, Belgrade, Serbia V. Milosevic Clinic of Neurology, Clinical Centre Nis, Nis, Serbia

blood samples prior to and 12 months after etanercept therapy. Plasma MMP-9 level was determined using an enzyme-linked immunosorbent assay kit. Clinical assessment was performed according to ACR Pedi 50 improvement criteria. The frequency of the A allele was significantly higher in JIA patients compared to controls (39% vs. 26%, P=0.026). Patients with the −308GG genotype achieved an ACR Pedi 50 response significantly more frequently than those with the −308AA genotype (P=0.035). MMP-9 level in patients with the genotype −308GG was significantly decreased after 1 year of treatment with etanercept compared to the value from before (P=0.036). On the other hand, there was a decrease of MMP-9 levels after treatment, but not statistically significant in patients with the genotypes −308GA/AA. We conclude that etanercept reduces MMP-9 level in children with polyarticular JIA and TNF-α-308GG genotype. Our results correlate with findings that the −308A allele is associated with a lower response to etanercept treatment. Keywords Etanercept . Juvenile idiopathic arthritis . MMP-9 . TNF-α-308 polymorphism

Introduction Juvenile idiopathic arthritis (JIA) is an inflammatory arthritis of unknown etiology that begins before the 16th birthday, persists in one or more joints for at least 6 weeks, and in which infectious arthritis and


other well-defined illnesses are excluded. Patients with JIA are divided into seven onset types, dependent on the number of joints involved and clinical signs and symptoms during the first 6 months of the illness. Polyarthritis affects five or more joints during the first 6 months of the disease and is divided into rheumatoid factor (RF)-positive and RF-negative polyarthritis [25]. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that are capable of degrading all major components of the extracellular matrix. The whole group can be divided into subclasses, such as collagenases, gelatinases, stromelysins, and membrane-type MMPs. MMP-9 belongs to the gelatinase subfamily (gelatinase B) of the MMPs, and therefore, its main substrate is gelatin (a denatured collagen). A number of studies have demonstrated that MMP-9 is an important mediator in inflammatory and connective tissue diseases and is thought to be one of the main mediators of joint damage in rheumatoid arthritis (RA) [5, 32, 34]. On the other hand, there are only few direct data on the activity of MMP-9 in JIA and its potential effect on cartilage destruction in the joints. Compelling evidence indicates that tumor necrosis factor-α (TNF-α) plays a central role in sustaining inflammatory process in joints of JIA patients [10, 13, 21, 28]. Moreover, TNF-α is a potent stimulator of mesenchymal cells, such as synovial fibroblasts, osteoclasts, and chondrocytes, which release tissuedestroying MMPs, including MMP-9 [5]. As TNF-α plays a central role in the inflammatory process, several strategies for the production of biological agents (etanercept, infliximab, adalimumab), designed to inhibit the activity of TNF-α, have been adopted. Etanercept is a fusion protein consisting of two identical chains of the recombinant extracellular TNF receptor II monomer fused with the Fc domain of human IgG1. Etanercept binds TNF-α and lymphotoxin and inhibits their activity. It has proven highly efficacious in children with JIA, especially those with polyarticular disease course [20]. Several single nucleotide polymorphisms (SNPs) have been identified in the TNF-α gene promoter [1]. Among these, a common polymorphism in the promoter, a guanine to adenine (G → A) substitution at position −308, has been studied intensively. It is not clear whether the TNF-α promoter −308G/A polymorphism has a functional significance, but there are

J. Basic et al.

some indications that the A allele could be associated with greater levels of TNF-α transcription [2, 19, 33]. Although some authors suggest that G → A point mutation in the promoter region has been associated with RA and systemic lupus erythematosus [8, 27], it is still unclear whether this SNP is associated with susceptibility to JIA. Also, there are no data showing the interdependence of this polymorphism and MMP9 level. The aim of this study was to investigate the distribution of TNF-α-308G/A polymorphism in polyarticular JIA patients compared to controls, as well as to evaluate whether this SNP can influence MMP-9 level and etanercept treatment response of JIA patients.

Patients and methods Subjects and sample collection JIA was diagnosed according to International League of Associations for Rheumatology (ILAR) classification criteria [25]. Sixty six JIA patients, with active polyarticular disease course, defined by the presence of five or more joints with active arthritis, were enrolled. Joints were defined as active by the presence of swelling or, if no swelling was present, by the limitation of motion accompanied by pain, tenderness, or both. Patients had active disease despite standard disease modifying antirheumatic drugs (methotrexate 15–20 mg/m2/week) and steroid therapy. In order for patients to be eligible for treatment with etanercept, failure to respond or intolerance to methotrexate was required. Etanercept therapy was given at the dosage of 0.4 mg/kg subcutaneously twice a week. All JIA patients were included in the etanercept national registry survey, willing to donate paired blood samples before starting etanercept therapy and 12 months afterward. Clinical response to etanercept was assessed after 12 months of therapy according to American College of Rheumatology Pediatric 50% (ACR Pedi 50) improvement criteria, using six core variables: the physician global assessment of disease activity, on a 10-cm visual analog scale (VAS; 0– 100); the parent/patient global assessment of overall wellbeing, on a 10-cm VAS (0–100); the Childhood Health Assessment Questionnaire (CHAQ; 0 -3); the number of joints with limited range of motions (LOM); and the number of joints with active arthritis

TNF-α-308 polymorphism and MMP-9 in JIA

(AA); and the erythrocyte sedimentation rate. ACR Pedi 50 improvement score was defined by international consensus: three or more JIA criteria improved by at least 50% with respect to baseline and no more than one core set criteria worsened by 30% [14]. The control group consisted of 65 healthy children, matched by age and sex with the patients. Venous blood samples were obtained from the median cubital vein and collected into EDTA vacutainer tubes. Two-hundred microliters of blood were used for DNA isolation. All samples were immediately centrifuged at 2,000×g for 15 min at 4°C afterward. Plasma was carefully removed and stored at −80°C until analysis. The present study was approved by the Ethical Committee of the Medical Faculty, University of Nis, Serbia. The relevant patients’ information and informed consent were provided in both written and oral form and signed by parents or by the patients if they were aged ≥18 years.


The presence of the A allele resulted in an uncut, full-length product (117 bp), while the presence of the G allele was detected as fragments of 97 and 20 bp on the gel pattern obtained. MMP-9 measurement Plasma total MMP-9 level was determined using the sandwich enzyme-linked immunosorbent assay (ELISA; Amersham Biosciences, Little Chalfont, UK) according to the manufacturer’s instructions. MMP-9 concentration was expressed as nanograms per milliliter. The sensitivity of the assay was 0.6 ng/ ml. The intra-assay and inter-assay CV were 2.3% and 7.5%, respectively. Minimum detectable dose was less than 0.156 ng/ml. According to the manufacturer’s brochure, no significant cross-reactivity or interference with various proteins, including mutual cross-reactivity between MMP-9, was observed. Statistical analysis

DNA analysis Genomic DNA was isolated from the whole blood samples using QIAamp DNA Blood Mini Kit (Quiagen GmbH, Hilden, Germany). The −308G/A polymorphism was determined by the polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) technique. A fragment of 117 bp was amplified using the forward primer 5′-AGGCAA TAGGTTTTGAGGGCCAT-3′ and the reverse primer 5′-ACACTCCCCATCCTCCCTGCT-3′. PCR was performed in a final volume of 25 μl containing 20 ng of DNA, 5 μl of Q solution, 2.5 μl of 10× PCR buffer, 10 mM of dNTP, 20 pmol of each primer, and 0.5 U of HotStarTaq DNA Polymerase (Qiagen GmbH, Hilden, Germany). The PCR conditions were as follows: initial denaturation at 95°C for 15 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min, ending with the final extension at 72°C for 10 min. PCR products were electrophoresed through 2% agarose gel, stained with ethidium bromide and visualized under UV light. The amplification products were digested with the restriction enzyme NcoI (Fermentas GmbH, St. Leon-Rot, Germany) at 37°C overnight and analyzed by 8% polyacrylamide gel electrophoresis. Gel was stained in ethidium bromide and visualized under UV light.

The allele and genotype frequencies were determined in the patients and controls and were compared with the values predicted by the Hardy–Weinberg equilibrium by means of the χ2 test. We performed a χ2 test to compare TNF polymorphism with clinical response after 12 months (ACR Pedi 50). Results for MMP-9 level were expressed as the mean ± SD. The difference between JIA patient’s and control’s means was verified by using an unpaired Student t test. The statistical comparison of data from JIA patients before and after 12 months’ treatment with etanercept was performed using a paired Student t test. Values of P< 0.05 were taken as statistically significant. Statistical analyses were performed using the SPSS version 13.0 statistical package (SPSS Inc., Chicago, IL, USA).

Results JIA patients population consisted of 45 girls (68.2%) and 21 boys (31.8%) with the mean age at disease onset 9.60 ± 3.89 years, disease duration 4.80 ± 2.79 years, and age at the study entry 14.36± 3.43 years (range 4–25, diagnosed before 16 years). In the control group of 65 healthy children, there were 38 girls (58.5%) and 27 boys (41.5%) with the mean age at the study entry 13.53±3.19 years. No signif-

176 Table 1 Genotype frequencies of the TNF-α promoter −308G/A polymorphism in children with polyarticular JIA and controls df degree of freedom

J. Basic et al. Genotype

Polyarticular JIA (n=66)

Controls (n=65)


19 (28.8%)

37 (56.9%)


42 (63.6%)

22 (33.8%)


5 (7.6%)

6 (9.3%)

icant differences were seen between demographic characteristics of the patients and controls. All patients had polyarticular disease course and only six of them were RF positive. Genotype frequencies for SNP were in Hardy– Weinberg equilibrium both in the patients (P= 0.165) and controls (P= 0.866). The genotype −308GG was present in 19 (28.8%) JIA patients, and the −308AA genotype was present in five (7.6%) patients. Forty-two (63.6%) patients were heterozygous (−308GA). The genotype frequency distributions of the TNF-α-308 polymorphism in the patients were significantly different from those of the controls (χ2 = 12.120; P= 0.002; Table 1). The frequency of the A allele was significantly higher in JIA patients compared to controls (39% vs. 26%; χ2 = 5.207; P= 0.026; Table 2). The clinical response to etanercept was analyzed by calculating ACR Pedi 50 response at 12 months in 50 of 66 patients. Those patients, who achieved an ACR Pedi 50 response, were regarded as responders. At month 12, 93.3% of patients with the genotype −308GG achieved an ACR Pedi 50 response, whereas 87.1% of patients with the genotype −308GA and 50.0% of patients with the genotype AA achieved an ACR 50 response. Patients with the −308GG genotype achieved ACR Pedi 50 response significantly more frequent than those with the −308AA genotype (χ2 =4.460, P=0.035). Patients with the genotype −308GG more frequently responded to etanercept treatment than patients who had the −308GA/AA genotype (78.78%), but this observation did not reach significance (χ2 =2.381, P=0.246; Table 3). Four of 50 patients (8.0%) achieved an ACR Pedi 30 response (≥30% improvement in three of six core variables, and no more than one core set criteria worsened by Table 2 Allele frequencies of the TNF-α promoter −308G/A polymorphism in children with polyarticular JIA and controls


P value (df=2; χ2 test)


30%) and were regarded as patients with low response to treatment, while three out of 50 patients (6.0%) did not respond to therapy. Out of 66 patients, only 50 have donated paired blood samples for MMP-9 level evaluation. The levels of MMP-9 in patients with JIA, either before (319.73±211.25 ng/ml) or after treatment (255.20± 172.13 ng/ml), were increased compared to control (27.96±15.88 ng/ml; P

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