Matrix metalloproteinase–9 promoter polymorphisms in Korean patients with systemic lupus erythematosus

Human Immunology (2008) 69, 374 –379

Matrix metalloproteinase–9 promoter polymorphisms in Korean patients with systemic lupus erythematosus Yun Jong Leea, Mijung Woob, Jung-Hyun Namb, Jinah Baekb, Churl Hyun Ima, Eun Young Leea, Eun Bong Leea, Kyung Sook Parkb, Yeong Wook Songa,* a b

Department of Internal Medicine, Medical Research Center, Seoul National University College of Medicine, Seoul, Korea Department of Biology, Sungshin Women’s University, Seoul, Korea

Received 2 December 2007; received in revised form 26 March 2008; accepted 28 March 2008

KEYWORDS Matrix metalloproteinase–9; Polymorphism; Systemic lupus erythematosus

Summary To investigate the association between functional promoter polymorphisms of matrix metalloproteinase–9 (MMP-9) and systemic lupus erythematosus (SLE), we analyzed MMP-9 promoter -1562 C⬎T and MMP-9 -90 (CA)n repeat polymorphisms in 135 Korean SLE patients (mean age, 34.7 years; 124 female and 11 male) and in 135 gender- and age-matched healthy controls (mean age, 35.4 years). Clinical and laboratory findings were collected during the follow-up period (mean, 63.5 months; range, 3–252 months), and Systemic Lupus International Collaborating Clinics/American College of Rheumatology (SLICC/ACR) Damage Indexes were calculated. The levels of total MMP-9 were measured in sera of SLE patients and controls by enzyme-linked immunoabsorbent assay. The serum levels of MMP-9 in SLE patients were significantly lower than those of controls (mean ⫾ standard error of the mean, 1421.6 ⫾ 177.4 vs 3731.4 ⫾ 441.4 ng/ml, p ⫽ 1.2⫻10⫺5 by t test). Both functional polymorphisms were under the Hardy-Weinberg equilibrium state except (CA)n repeat polymorphisms in SLE patients (p ⫽ 2.6⫻10⫺5 by ␹2 goodness-of-fit test). The distribution of the MMP-9 promoter polymorphisms or haplotypes was not significantly different in SLE patients and controls. However the frequency of alleles with low numbers of CA repeats (n ⬍ 21, 11.9% vs 7.0%, p ⫽ 0.06 by the ␹2 test; odds ratio ⫽ 1.78, 95% confidence interval ⫽ 0.99⫺3.20) and the prevalence of low CA repeats homozygote tended to be higher in patients than in controls (5.2% vs 0.7%, p ⫽ 0.07 by logistic regression, odds ratio ⫽ 7.29, 95% confidence interval ⫽ 0.88⫺60.10) in the recessive model. No relationship was found between MMP-9 polymorphisms and clinical features or damage as indicated by SLICC/ACR Damage Index in the study subjects. These results suggest that genetic polymorphisms of the MMP-9 promoter regions are not associated with the development of SLE in Korea. © 2008 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

* Corresponding author. Fax: 822-762-9662. E-mail address: [email protected] (Y.W. Song).

0198-8859/$ -see front matter © 2008 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2008.03.005

375

MMP-9 polymorphism in SLE

Introduction Matrix metalloproteinases (MMPs) participate primarily in the degradation and remodeling of the extracellular matrix and are associated with a variety of physiologic and pathologic conditions such as cancer invasion and metastasis, joint destruction in arthritis, and atherosclerotic vascular diseases. Gelatinolytic MMP-9 helps immune cells to migrate through cleaved basement membrane and can generate autoimmune remnant neo-epitopes. In addition it may play a regulatory role in immune response because it activates proform proteins or truncates active forms [1,2]. There is some evidence suggesting that MMP-9 have a role in the pathogenesis of systemic lupus erythematosus (SLE). It was reported that the expression of MMP-9 was upregulated in the inflamed tissue and it was highly secreted from peripheral blood mononuclear cells in murine lupus models and SLE patients [3–10]. The transcriptional activity of MMP-9 is known to be affected by the promoter polymorphisms [11,12], but no study has been conducted on MMP-9 genetic polymorphisms in SLE patients. We therefore studied the functional polymorphisms of MMP-9 promoter, MMP-9 -1562C⬎T (SNP ID rs3918242) and CA dinucleotide repeats polymorphism (SNP ID rs3080277) in Korean patients with SLE.

Subjects and methods Study subjects

-1562C⬎T polymorphisms was performed using the polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) method, as described previously [15]. The primers were 5’GCCTGGCACATAGTAGGCCC-3’ (forward) and 5’-CTTCCTAGCCAGCCGGCATC-3’ (reverse), and a restriction enzyme SphI was used to digest the PCR products. For genotyping the MMP-9 microsatellite polymorphism, we adapted the method described by Maeda et al. [16] with some modification. After amplification with the primers (forward, 5’-GACTTGGCAGTGGAGACTGCGGGCA-3’; reverse, 5’GACCCCACC- CCTCCTTGACAGGCAA-3’), PCR amplicons were analyzed by 6% urea-denatured acrylamide gel electrophoresis and silver staining.

Statistical analysis The ␹2 goodness-of-fit testing was used to determine whether the genotype frequencies deviated from Hardy-Weinberg equilibrium in controls and patients separately. The association of the genotypes with SLE was tested using binary logistic regression analysis under co-dominant, dominant, or recessive model and the homozygote for allele with a high promoter activity (i.e., T allele or alleles with the number of CA repeats ⱖ21) [11] was used as a reference. Results were expressed as odds ratios (ORs) with 95% confidence intervals (CIs). The G*power program was used to calculate the statistical power (http://www.psycho.uni-duesseldorf.de/abteilungen/aap/ gpower3). Haplotype frequencies of MMP-9 promoter gene were computed and then the differences in the haplotype frequencies between SLE patient and control groups were analyzed by permutation tests (100,000 random permutations) using PHASE program version 2.1 [17]. To study the associations between genetic polymorphisms and clinical features, SLE patients were subgrouped according to genotypes, serologic status, or clinical manifestations shown in Table 1. Comparisons of dichotomous variables were performed using the ␹2 or Fisher’s exact test, as appropriate. Mean differences between continuous variables were evaluated using the t test or Mann-Whitney test. All statistical analyses were performed using SPSS for Windows, version 11.0.1 (SPSS Inc., Chicago, IL).

In this study, 135 SLE patients (mean age, 34.7 years; range, 19 –75 years; 124 female and 11 male) and 135 healthy controls (mean age, 35.4 years; range, 20 –72 years) and were recruited from Seoul National University Hospital. Controls were individually gender- and age-matched (range ⫾ 3 years) healthy persons who were found to have no specific diseases at a medical checkup. SLE was diagnosed according to the criteria proposed by the American College of Rheumatology (ACR) in 1997 [13]. Clinical and laboratory findings were defined using ACR criteria and collected from well-documented medical records during the follow-up period (mean, 63.5 months; range, 3–252 months). SLICC/ACR (Systemic Lupus International Collaborating Clinics/American College of Rheumatology) Damage Index was calculated at the time of blood sampling (mean, 1.4; range, 0 –7) [14]. Of the 72 SLE patients with a history of proteinuria greater than 500 mg/day, 34 patients underwent renal biopsy and 27 (79.4%) had a diffuse or focal proliferative lesion. Neuropsychiatric manifestations in 15 patients (11.2%) included cerebrovascular disease (three events), headache (one event), movement disorder (one event), seizure disorders (three events), acute confusional state (two events), mood disorder (one event), psychosis (six events), and cranial neuropathy (one event). The demographic and clinical features of the SLE group are summarized in Table 1.

The levels of serum MMP-9 in patients with SLE was significantly lower than those of controls (mean ⫾ SEM, 1421.6 ⫾ 177.4 vs 3731.4 ⫾ 441.4 ng/ml, p ⫽ 1.2⫻10⫺5 by t test; Figure 1). In SLE patients, patients with malar rash had lower concentration of serum MMP-9 than those without malar rash (1127.8 ⫾ 240.1 vs 1698.9 ⫾ 254.7 ng/ml, p ⫽ 0.03 by Mann-Whitney test). The levels of MMP-9 were significantly positively correlated with the numbers of WBC (p ⫽ 0.001, Spearman’s coefficient ⫽ 0.55) or neutrophil (p ⫽ 0.003, Spearman’s coefficient ⫽ 0.51).

Measurement of serum MMP-9

Functional MMP-9 promoter polymorphisms

Because sera were not available for all subjects, 40 SLE patients (33 female and seven male) and 40 controls were randomly selected. The serum concentrations of total MMP-9 were determined using a commercial enzyme-linked immunoabsorbent assay (ELISA; R&D Systems, Inc., Minneapolis, MN).

The genotype frequencies of -1562 C⬎T in controls or SLE patients did not depart significantly from Hardy-Weinberg equilibrium (p ⫽ 0.68 and p ⫽ 0.46 by ␹2 goodness-of-fit test, respectively). In the co-dominant, dominant, and recessive models, there was no skewed distribution of -1562 C⬎T polymorphisms of MMP-9 in SLE patients compared with those of controls (Table 2). In terms of the (CA)n repeat polymorphisms of MMP-9, repeat numbers ranged from 14 to 24 in SLE patients and

DNA isolation and genotyping Genomic DNA was extracted from peripheral blood using QIAamp DNA Blood kits (Qiagen, Valencia, CA). Genotyping for MMP-9

Results Serum levels of total MMP-9

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Y.J. Lee et al.

ABBREVIATIONS ACR MMP-9 PCR-RFLP SLE SLICC/ACR

American College of Rheumatology matrix metalloproteinase–9 polymerase chain reaction–restriction fragment length polymorphism systemic lupus erythematosus Systemic Lupus International Collaborating Clinics/American College of Rheumatology

from 18 to 24 in controls (Figure 2). The most frequent allele was (CA)21 in both groups (45.2% in patients and 51.9% in controls), and the most frequent genotype was (CA)21/ (CA)23 (31.9% in patients and 30.4% in controls). When the (CA)n repeats of MMP-9 promoter polymorphisms were stratified according to the promoter activity, that is, categorized as high if n of (CA)n was ⱖ21 and low if n of (CA)n was ⬍21, the genotype frequencies were in Hardy-Weinberg equilibrium in control individuals (p ⫽ 0.66 by the ␹2 goodnessof-fit test), but those in SLE patients were not in HardyWeinberg equilibrium (p ⫽ 2.6⫻10⫺5). The frequency of alleles with low numbers of CA repeats (i.e., allele “a” in

Table 1 Characteristics of patients with systemic lupus erythematosus (n ⫽ 135). Age (y) Female:male ratio Duration of disease (mo) No. of ACR criteria satisfied SLICC/ACR Damage Index Clinical featuresb Oral ulcer Discoid rash Malar rash Photosensitivity Arthritis Serositis Neuropsychiatric lupus Laboratory findingsb Leukopenia Hemolytic anemia Thrombocytopenia Proteinuria Proliferative lupus nephritis Antinuclear antibody Anti-dsDNA Anti-Sm Anti-phospholipid

34.7 ⫾ 1.0 (75–19)a 124:11 63.5 ⫾ 3.9 (252–3) 5.5 ⫾ 0.1 (4–9) 1.4 ⫾ 0.1 (0–7) 51 6 71 35 88 43 15

(37.8%) (4.4%) (52.6%) (25.9%) (65.2%) (31.9%) (11.2%)

78 42 37 72 27 127 110 16 19

(57.8%) (31.1%) (27.4%) (53.3%) (79.4%c) (94.1%) (81.5%) (47.1%d) (28.4%e)

ACR, American College of Rheumatology; Systemic Lupus International Collaborating Clinics/American College of Rheumatology. a Mean ⫾ SEM (range). b Number of patients with corresponding event(s) during their courses. c Among 34 patients who underwent renal biopsy. d Among 34 patients with available data. e Among 67 patients with available data.

Figure 1. Serum matrix metalloproteinase–9 (MMP-9) concentrations in controls and systemic lupus erythematosus (SLE) patients.

case of n ⬍21) tended to be higher in patients than in controls (11.9% vs 7.0%, p ⫽ 0.06 by ␹2 test; OR ⫽ 1.78, 95% CI ⫽ 0.99 –3.20). In addition, the prevalence of homozygote for low CA repeats alleles tended to be higher in patients than in controls (5.2% vs 0.7%, p ⫽ 0.07, OR ⫽ 7.29, 95% CI ⫽ 0.88 – 60.10) in the recessive model (Table 2). Among haplotypes reconstructed by the PHASE software, the haplotype C/b was most common in both control and patient groups, with “b” representing the number of (CA)n ⱖ21 (83.2% in controls and 78.6% in patients). Haplotype analysis did not show significantly different distributions between controls and SLE patients (p ⫽ 0.078 by permutation test). The haplotype for alleles with a higher promoter activity (i.e., haplotype T/b; p ⫽ 0.79, OR ⫽ 0.92, 95% CI ⫽ 0.48 –1.74) or with a lower promoter activity (i.e., haplotype C/a; p ⫽ 0.44, OR ⫽ 1.33, 95% CI ⫽ 0.65–2.76) did not increase the risk of SLE. Subgroup analyses according to serologic status or clinical manifestations, including proliferative nephritis and neuropsychiatric lupus, showed no significant differences in the distributions of the MMP-9 polymorphisms. In addition, according to MMP-9 genotypes, no significant difference was observed in terms of the age at onset, titer of anti-dsDNA, amount of proteinuria, number of ACR SLE criteria met, and SLICC/ACR Damage Index.

Discussion Many studies have addressed the issue of MMP expression in the blood and tissues of individuals with autoimmune inflammatory diseases. Among the MMPs, MMP-9 has been most frequently studied in SLE. Fresh peripheral blood mononuclear cells from lupus patients was observed to spontaneously secrete higher levels of MMP-9 [3]. In addition, increased MMP-9 expression was found in the cerebrospinal fluids [4,5], peripheral nerve [6], kidney [7–9], and synovial fluid [10] of individuals with SLE. It has been well established

377

MMP-9 polymorphism in SLE Table 2 Genotype distributions of matrix metalloproteinase–9 (MMP-9) promoter -1562 C/T and -90(CA)n repeat polymorphisms in control individuals and patients with systemic lupus erythematosus (SLE). Model MMP9 -1562 C⬎T Co-dominant model

Dominant model Recessive model MMP9 -90 (CA)n Co-dominant model

Dominant model Recessive model

Allele

Controls (n ⫽ 135)

SLE (n ⫽ 135)

OR (95% CI)

pa

T/T C/T C/C T/T C/T and C/C T/T and C/T C/C

1 26 108 1 134 27 108

(0.7%) (19.3%) (80.0%) (0.7%) (99.3%) (20.0%) (80.0%)

1 30 104 1 134 31 104

(0.7%) (22.2%) (77.0%) (0.7%) (99.3%) (23.0%) (77.0%)

— 1.25 1.03 — 1.06 — 0.83

0.81 0.88 0.99 0.97

b/b a/b a/a b/b a/b and a/a b/b and a/b a/a

117 17 1 117 18 134 1

(86.7%) (12.6%) (0.7%) (86.7%) (13.3%) (99.3%) (0.7%)

110 180 7 110 25 128 7

(81.5%) (13.3%) (5.2%) (81.5%) (18.5%) (94.8%) (5.2%)

— 1.14 7.40 — 1.49 — 7.30

(0.07–21.26) (0.06–16.78) (0.07–17.33)

0.53 (0.46–1.49)

(0.56–2.32) (0.90–61.20)

0.17 0.73 0.06 0.24

(0.77–2.88) 0.07 (0.88–60.10)

Allele a, allele with a CA repeat number of ⬍21; allele b, allele with a CA repeat number of ⱖ21; CI, confidence interval; OR, odds ratio. a Gender- and age-adjusted p value.

that the promoter activities of MMP-9 are influenced by the presence of polymorphisms. In vitro transfection assays showed that MMP-9 promoters containing fewer than 21 (CA) repeats or -1562C had lower transcriptional activities [11,12]. MMP-9 promoter polymorphisms have been reported to be associated with some diseases, in which matrix degradation by MMP-9 is considered to be important in the pathogenesis [15,16,18 –23]. Low MMP-9 production in itself, however, has been reported to be a potential genetic risk factor related to abnormally increased immune reactions and MMP-9 genotype(s) with a lower promoter activity may confer the susceptibility to an autoimmune disease. Several studies indicate that MMP-9 plays a regulatory role in immune response, e.g., MMP-9 regulates the activities of various cytokines and che-

Figure 2. Distribution of genotypes for the (CA)n repeats polymorphisms of matrix metalloproteinase–9 (MMP-9) promoter. Filled black bars represent number of systemic lupus erythematosus (SLE) patients with a corresponding genotype; white bars represent mean number of controls.

mokines via enzymatic proteolysis; potentiates the activities of pro–IL-1␤ and IL-8, and inhibits GRO␣ and IL-2R␣ [24,25]. Moreover MMP-9 released from cancer cells cleaves IL-2R␣, which may explain the immunosuppressed state in cancer patients, because IL-2R plays a pivotal role in the development and propagation of functional T cells [26]. Furthermore MMP-9 deficiency exhibited prolonged contact hypersensitivity in response to dinitrofluorobenzene [27], exacerbated functional and histologic glomerular injury in an antiglomerular basement membrane nephritis model [28], enhanced allergen-induced airway inflammation [29], and enhanced T-cell responsiveness and dendritic cell stimulatory capacity [30]. In this study there was no significant difference in the frequencies of both MMP-9 functional promoter polymorphisms. The prevalence of the MMP-9 genotype homozygous for low numbers of CA repeats tended to be higher in SLE patients than in controls (5.2% vs 0.7%), and the serum concentrations of total MMP-9 were significantly lower in the patients than in the controls. In contrast to its higher expression in the target organs [3–10] and increased activity in the serum [31], we observed that SLE patients had the lower MMP-9 concentrations in the sera than did controls. Our results were in line with previous studies concerning the levels of serum total MMP-9 in SLE [32–34]. The commercial ELISA kits used in this study is designed to measure both pro- and active MMP-9 levels, not gelatinolytic activity. Although the causes of lower MMP-9 serum levels have not been defined in SLE, the total serum concentrations can be affected by nongenetic factors such as sample collection methods [35] or number of neutrophils in peripheral blood, a major source of serum MMP-9 [36]. In our SLE patients, significant correlations were found between MMP-9 concentrations and WBC or neutrophil counts; therefore the present findings regarding serum MMP-9 levels cannot be explained only by a higher prevalence of homozy-

378 gote for low numbers of CA repeats in SLE and should be interpreted with caution. Demacq et al. reported that the serum MMP-9 levels were not dependent on their genetic polymorphisms [37]. The statistical power was not sufficient (45.6% for a type I error of 5%) in the present study because of small number of lower CA repeats homozygote in study subjects. The distributions of (CA)n repeats polymorphisms were reported differently according to race/ethnicity. In published papers showing detailed information on MMP-9 promoter microsatellite polymorphisms, the major allele of healthy control individuals in United States (51%), Italy (60%), and the United Kingdom (54%) is (CA)14 [18,19,38]. However most in Japan (42%) and Korea (49%) the major allele in healthy control individuals was (CA)21 [17,23]. In the present study, the frequency of (CA)n alleles with n ⱖ 21 was 93% in healthy controls, and the most common allele was (CA)21 (52%). Therefore the effects on the MMP-9 CA dinucleotide microsatellite polymorphisms need to be reevaluated in a larger cohort of individuals of multiple races/ ethnicities. In conclusion, although the prevalence of MMP-9 genotypes with low numbers of CA repeats (i.e., with lower activity) tended to be higher and the serum concentrations of MMP-9 were significantly lower in SLE patients in Korea, we did not find evidence that MMP-9 promoter polymorphisms confer genetic susceptibility to SLE.

Y.J. Lee et al.

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Acknowledgment [16]

This work was supported by a grant from the Korean Ministry of Science & Technology through the National Research Laboratory Program for Rheumatic Disease. [17]

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