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Rheumatology 2008 47(1):41-44; doi:10.1093/rheumatology/kem312
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Effect of matrix metalloproteinase-3 functional SNP on serum matrix metalloproteinase-3 level and outcome measures in Japanese RA patients

S. Tsukahara*, M. Shinozaki*, K. Ikari, T. Mochizuki, E. Inoue, T. Tomatsu, M. Hara, H. Yamanaka, N. Kamatani and S. Momohara

Institute of Rheumatology, Tokyo Women's Medical University, Tokyo, Japan.

Correspondence to: K. Ikari, Institute of Rheumatology, Tokyo Women's Medical University, 10-22 Kawada, Shinjuku, Tokyo 162-0054, Japan. E-mail: kikari{at}ior.twmu.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objective. A bi-allelic polymorphism on the promoter region, –1612 ins/del A, was found to influence the production of MMP-3. Since MMP-3 plays a particularly pivotal role in joint destruction, the MMP-3 gene is thought to be an interesting target gene of disease severity in RA. We attempt to determine whether the MMP-3 promoter polymorphism is associated with serum titre of MMP-3, disease activity and severity in Japanese RA patients.

Methods. DNA samples were obtained from 1504 RA patients as part of the Institute of Rheumatology Rheumatoid Arthritis observational cohort study. From the 2006 spring data, serum MMP-3 levels of 820 patients were available by enzyme immunoassay. Joint damage score at 5-yr disease duration could be measured using the Sharp/van der Heijde method in 162 patients. Genotyping of –1612 ins/del A was performed using fluorescent-labelled fragment analysis. Differences in serum MMP-3 level and joint damage score among genotypes of –1612 ins/del A polymorphism were analysed by linear regression analysis.

Results. No significant differences were found among MMP-3 genotypes on patient characteristics including disease activity score (P = 0.51) or health assessment questionnaire (P = 0.99). A significant effect of risk allele on serum MMP-3 level was observed (P = 0.038), while no significant effect was observed on radiographic joint damage (P = 0.47).

Conclusion. We conclude that MMP-3 functional polymorphism is associated with serum MMP-3 titre, but is not a direct predictor for outcome measures in Japanese RA patients.

KEY WORDS: Rheumatoid arthritis, MMP-3, Association, Gene, IORRA


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The MMPs are a family of zinc-dependent enzymes that degrade the components of the extracellular matrix (ECM) and are essential for tissue remodelling and repair during development and inflammation [1]. MMP-3 is the main metalloproteinase that is secreted by fibroblasts, synovial cells and chondrocytes. It degrades proteoglycans, fibronectin, laminin and type IV collagen, and moreover it activates other MMPs that synergistically regulate the accumulation of ECM [1]. MMP-3 expression is regulated with the promoter responding to stimuli including growth factors and cytokines.

Recent studies have shown that MMP-3 is the most abundant MMP in RA. Serum MMP-3 is elevated in patients with RA both in early and established disease and is associated with disease activity and severity [2]. Measurement of MMP-3 is widely accepted as a clinically useful method for evaluating disease activity of RA. Therefore, the MMP-3 gene is thought to be an interesting genetic component of disease activity and severity for RA.

A bi-allelic polymorphism in the promoter region, –1612 ins/del A (rs3025058), has been found to influence the production of MMP-3 [3]. One allele has a run of six adenosines (6A) and the other has five (5A), and the 6A allele has about half the promoter strength of the 5A allele [3]. Recent studies in European populations indicate that this promoter polymorphism is associated with the progression of radiographic joint destruction of RA [4, 5]. They show that the 6A/6A genotype is associated with high radiographic damage scores. However, the disadvantageous genotype producing MMP-3 and its relation to the progression of joint destruction in RA is paradoxical.

In order to predict the disease activity and severity to improve therapeutic outcome, it is interesting to know whether this polymorphism is indeed associated with the disease activity and the progression of joint damage in diseased individuals. In this study, we conducted a test to find the association between the MMP-3 promoter 5A/6A polymorphism and the serum titre of MMP-3, disease activity and disease severity in patients from a large-scale Japanese RA cohort.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Tokyo Women's Medical University Genome Ethics Committee approved the present study and each individual signed an informed consent form after receiving a verbal explanation of the study. It is part of a large-scale Japanese RA cohort project, IORRA (Institute of Rheumatology Rheumatoid Arthritis cohort) [6]. The diagnosis of RA was based on the American College of Rheumatology 1987 revised criteria [7]. DNA samples were obtained from 1504 RA patients.

Genotyping of the 5A/6A polymorphism was performed using fluorescent-labelled fragment analysis. An extra sequence (GTTTCTT) was attached to the 5' end of the reverse primer to promote non-templated addition of adenine for accurate genotyping [8]. The PCR was performed in a 384-well plate in a final volume of 5 µl, containing 2 ng of genomic DNA, 0.25 mmol/l dNTPs, 0.5 µl 10 x PCR buffer (containing 15 mmol/l MgCl2), 1.5 pmol of each primer (5'-[6-FAM]-GAT TAC AGA CAT GGG TCA CG-3' and 5'-[GTTTCTT]-GAA TTC ACA TCA CTG CCA CC-3') and 0.2 U AmpliTaq Gold (Applied Biosystems, Tokyo, Japan). After a pre-PCR heating step for 12 min at 95°C, 35 cycles of amplification (15 s at 94°C for denaturing, 15 s at 55°C for annealing and 30 s at 72°C for extension), plus 10 min final extension, were performed in a Dual 384-Well GeneAmp PCR System (Applied Biosystems). After electrophoresing PCR products on an automated capillary DNA analyser (ABI Prism 3100, Applied Biosystems), DNA fragment size was determined with Genescan software and genotyping of the 5A/6A polymorphism was carried out with Genotyper software (Applied Biosystems).

In the spring of 2006, blood samples from 820 RA patients were collected, immediately centrifuged and stored. Serum MMP-3 levels were determined by EIA using monoclonal antibodies (Mitsubishi Kagaku Medience, Tokyo, Japan) [9]. The EIA system is capable of measuring both precursor and active forms of MMP-3. The demographic, clinical and treatment details of these patients in the spring of 2006 were obtained from the IORRA database, which includes the disease activity score (DAS) with 28 joints (DAS28) and the Japanese version of the health assessment questionnaire (Table 1) [10, 11].


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  		TABLE 1. Characteristics of patients

 
Radiographs of the hands and feet of the patients at 5-yr disease duration were obtained retrospectively, and radiographic joint damage was assessed using the Sharp/van der Heijde (SvdH) method [12].

The chi-squared test was used to compare the observed numbers of each genotype with those expected for a population in Hardy–Weinberg equilibrium. Differences in patient characteristics among MMP-3 genotypes were assessed with Kruskal–Wallis test or Fisher's exact test. The allelic effect of 5A/6A polymorphism on serum MMP-3 level and SvdH score was analysed by linear regression analysis. These tests were implemented in the R software package, version 2.4.1 (http://www.r-project.org/).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
The serum MMP-3 level of the patients with genotype data could be detected in 812 patients from the 2006 spring data. SvdH score at 5-yr disease duration could be measured in 162 patients. The observed genotype frequencies of the polymorphism were in Hardy–Weinberg equilibrium. No significant differences were found among MMP-3 genotypes in patient characteristics including DAS (P = 0.76) or health assessment questionnaire (P = 0.99, Table 1). Significant differences were found among MMP-3 genotypes for serum MMP-3 level [P = 0.038; the medians were 30.9, 66.4 and 78.9, respectively, for genotypes 5A/5A (n = 19), 5A/6A (n = 186) and 6A/6A (n = 517); Fig. 1A]. Though a trend of an effect of 5A allele on SvdH score was observed, it was not significant [P = 0.47; the medians were 66, 50 and 39, respectively, for genotype 5A/5A (n = 2), 5A/6A (n = 35) and 6A/6A (n = 125); Fig. 1B].


Figure 1
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  		FIG. 1. Each boxplot shows the different –1612 ins/del A genotypes for (A) serum titre of MMP-3 and (B) Sharp/van der Heijde score. Each box represents the interquartile range of values, with the bold line showing the median value. The vertical lines show maximum and minimum value that fall within 1.5 box lengths, the open circles show extreme values >1.5 box plot lengths.

 

   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
Predicting the disease course of RA may allow better targeting of aggressive treatment to improve therapeutic outcome. Since serum MMP-3 is associated with disease activity and severity of RA, the 5A/6A polymorphism of the promoter region has been of considerable interest related to the disease outcome of RA.

In the present study, while we found an additive effect of the 6A allele on increasing level of serum MMP-3, no significant effect of the polymorphism was found on the disease activity or severity of RA in Japanese patients. Although the former result was similar to that of a recent in vivo study that showed an association between higher serum MMP-3 level and the 6A/6A genotype [5], the result was inconsistent with the previous report of association between the 6A allele and lower transcriptional activity in vitro [3] (Table 2). On the contrary, though we confirmed the result of the previous study using Caucasian samples that showed no association of MMP-3 genotypes and DAS [13], we could not replicate the relation of MMP-3 genotypes to the progression of joint destruction that the European studies have indicated recently [4, 5].


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  		TABLE 2. Characteristics of patients and results of analyses in previous studies

 
There are several possible explanations for the discrepancies. The simplest explanation is that they may be due to an ethnicity-specific effect. The allelic effect of 6A on MMP-3 titre may exist in the Japanese population; however, an effect that is smaller than in Caucasians might have an impact on progressive joint damage. It is not unusual to find ethnic differences in genetic studies [14]. Further studies using independent Japanese population samples will be helpful to establish the association.

A possible reason for the failure in replicating association between the genotypes and the joint damage score is lack of statistical power. While the allelic effect of the 5A/6A polymorphism on the serum MMP-3 level was tested using 812 patients, the effect on radiographic progression in the first 5 yrs was tested only in 162 patients due to the inconsistency of radiographs at 5-yr disease duration for many of the samples. Reasons for the unavailability for proper radiographs were because the disease duration of some patients was <5 yrs, patients were included in the cohort after >5 yrs of disease duration, or some did not have the appropriate set of radiographs taken at any time during the designated period. It is expected that an increase in the number of DNA samples with radiographic data is necessary to obtain more statistical power.

Another possible reason for the failure of genotypical association and joint damage score is because we used the samples at 5-yr disease duration for the study of joint damage. The previous Japanese study indicated that the level of serum MMP-3 is associated with the joint damage score in early RA patients [2].

A potential explanation for the paradox that the disadvantageous genotype to produce the protein in vitro is related to the higher serum MMP-3 level in RA patients, which confirmed the recent European studies that the higher MMP-3 titre is associated with the 6A allele, is that genotype-related expression is different for the different cell types. It might be necessary to examine the effect on the genotype-related expression in cell subsets involving pathology of RA for the post-translational modification of MMP-3 protein.

The modification of serum MMP-3 level in patients by their treatments could put a bias on the results and might also cause the discrepancies. However, differences regarding medication usage of NSAIDs, DMARDs, biologicals or steroids among the MMP-3 genotypes could not be observed.

Recent reports have found that the effect of MMP-3 polymorphism on radiographic damage in RA was emphasized by HLA-DRB1 shared epitope alleles [4, 15]. It will be interesting to test the interaction between MMP-3 and HLA in Japanese RA patients, since the HLA locus is estimated to account for one-third of the genetic component of RA [16].

In conclusion, MMP-3 functional polymorphism is associated with serum MMP-3 titre in RA patients, but the effect is not strong enough to have an impact on disease activity or radiographic joint damage. The 5A/6A polymorphism might not be a prognostic factor of the disease outcome in Japanese RA patients.

Formula


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
We thank Dr Iikuni for her assistance in preparing the manuscript. We are also grateful to 36 pharmaceutical companies for supporting the IORRA cohort, and Prof. Taniguchi and other members of our institute for their efforts on IORRA.

Funding: This work was supported by a grant provided by the Japan Orthopaedics and Traumatology Foundation (to K.I.), Takeda Science Foundation (to K.I.) and a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (to K.I. and S.T.).

Disclosure statement: The authors have declared no conflicts of interest.


   Notes
 
*S. Tsukahara and M. Shinozaki contributed equally to this work. Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Okada Y. Proteinases and matrix degeneration. In: Kelley's textbook of rheumatology—Harris ED, Budd RC, Firestein GS, et al, eds. (2005:) 7th. Philadelphia: Elsevier Saunders. 63–81.
  2. Yamanaka H, Matsuda Y, Tanaka M, et al. Serum matrix metalloproteinase 3 as a predictor of the degree of joint destruction during the six months after measurement, in patients with early rheumatoid arthritis. Arthritis Rheum (2000) 43:852–8.[CrossRef][Web of Science][Medline]
  3. Ye S, Eriksson P, Hamsten A, Kurkinen M, Humphries SE, Henney AM. Progression of coronary atherosclerosis is associated with a common genetic variant of the human stromelysin-1 promoter which results in reduced gene expression. J Biol Chem (1996) 271:13055–60.[Abstract/Free Full Text]
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  5. Mattey DL, Nixon NB, Dawes PT, Ollier WE, Hajeer AH. Association of matrix metalloproteinase 3 promoter genotype with disease outcome in rheumatoid arthritis. Genes Immun (2004) 5:147–9.[CrossRef][Web of Science][Medline]
  6. Tanaka E, Saito A, Kamitsuji S, et al. Impact of shoulder, elbow, and knee joint involvement on assessment of rheumatoid arthritis using the American College of Rheumatology Core Data Set. Arthritis Rheum (2005) 53:864–71.[CrossRef][Web of Science][Medline]
  7. Arnett FC, Edworthy SM, Bloch DA, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum (1988) 31:315–24.[Web of Science][Medline]
  8. Brownstein MJ, Carpten JD, Smith JR. Modulation of non-templated nucleotide addition by Taq DNA polymerase: primer modifications that facilitate genotyping. Biotechniques (1996) 20:1004-6–1008-10.
  9. Obata K, Iwata K, Okada Y, et al. A one-step sandwich enzyme immunoassay for human matrix metalloproteinase 3 (stromelysin-1) using monoclonal antibodies. Clin Chim Acta (1992) 211:59–72.[CrossRef][Web of Science][Medline]
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  11. Prevoo ML, van't Hof MA, Kuper HH, van Leeuwen MA, van de Putte LB, van Riel PL. Modified disease activity scores that include twenty-eight-joint counts. Development and validation in a prospective longitudinal study of patients with rheumatoid arthritis. Arthritis Rheum (1995) 38:44–8.[Web of Science][Medline]
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Submitted 22 June 2007; revised version accepted 26 October 2007.
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