Molecular Biology Reports

, Volume 40, Issue 11, pp 6303–6308

MMP-2, TNF-α and NLRP1 polymorphisms in Chinese patients with ankylosing spondylitis and rheumatoid arthritis

Authors

  • Rongbin Sun
    • Department of Orthopedic TraumaAffiliated Hospital of Nanjing Medical University, Changzhou Second People’s Hospital
  • Yong Huang
    • Department of Orthopedic TraumaAffiliated Hospital of Nanjing Medical University, Changzhou Second People’s Hospital
  • Hui Zhang
    • Department of Orthopedic TraumaAffiliated Hospital of Nanjing Medical University, Changzhou Second People’s Hospital
    • Department of Orthopedic TraumaAffiliated Hospital of Nanjing Medical University, Changzhou Second People’s Hospital
    • Central LaboratoryAffiliated Hospital of Nanjing Medical University, Changzhou Second People’s Hospital
Article

DOI: 10.1007/s11033-013-2743-8

Cite this article as:
Sun, R., Huang, Y., Zhang, H. et al. Mol Biol Rep (2013) 40: 6303. doi:10.1007/s11033-013-2743-8

Abstract

Rheumatoid arthritis (RA) and ankylosing spondylitis (AS) are autoimmune, inflammatory diseases with substantial genetic contributions. Matrix metalloproteinase (MMP)-2, tumor necrosis factor (TNF)-α and NLR family pyrin domain-containing 1 (NLRP1) play important roles in the immune response. We studied the MMP-2 rs243865 C/T, TNF-α rs1800629 A/G, NLRP1 rs878329 C/G and NLRP1 rs6502867 C/T polymorphisms in a Chinese cohort of 520 patients with RA, 100 with AS and 520 controls. Genotyping was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Using the MMP-2 rs243865 CC homozygote genotype as the reference group, the CT and TT/CT genotypes were associated with significantly reduced risks of AS. However, logistic regression analyses revealed that the MMP-2 rs243865 C/T polymorphism was not associated with risk of RA. TNF-α rs1800629 A/G, NLRP1 rs878329 C/G and NLRP1 rs6502867 C/T polymorphisms were not associated with risk of RA or AS. These findings suggest that the MMP-2 rs243865 C/T polymorphism is associated with AS development.

Keywords

MMP-2PolymorphismAnkylosing spondylitisRheumatoid arthritisMolecular epidemiology

Abbreviations

CI

Confidence interval

LD

Linkage disequilibrium

MMP-2

Matrix metalloproteinase-2

NLRP1

NLR family pyrin domain-containing 1

OR

Odds ratio

SNP

Single nucleotide polymorphism

TNF-α

Tumor necrosis factor-α

Introduction

Rheumatoid arthritis (RA) and ankylosing spondylitis (AS) are autoimmune, inflammatory diseases with substantial genetic contributions [1, 2]. Matrix metalloproteinases (MMPs) play important roles in RA and participate in the development of RA-associated cartilage and bone damage [3]. MMP-2, also known as gelatinase, is involved in inflammation and immunity, as well as degradation and remodeling of the extracellular matrix [4]. MMP-2 expression is up-regulated in many inflammatory and immune diseases [5] and is overproduced in the joints of RA patients, where it plays a key role in articular cartilage degradation [6, 7]. Among the metalloproteinase genes, MMP-2 showed the highest expression levels in a microarray study in synoviocytes [8]. Itoh et al. demonstrated that overexpression of MMP-2 played a suppressive role in the development of inflammatory joint disease [9]. MMP-2 affects the clearance of recruited immune cells, forms part of an interleukin 13 (IL-13)-dependent regulatory loop, and is necessary for resolving inflammatory reactions [10]. IL-17A has been shown to upregulate the expression of MMP-2 at the transcriptional and translational levels [11], while IL-17A and tumor necrosis factor-α (TNF-α) upregulated the expression of MMP-2 in normoxic RA-fibroblast-like synoviocytes [8]. The MMP-2 gene is located on chromosome 16q13, with 13 exons and 12 introns. The MMP-2 −1306 C/T (rs243865) single nucleotide polymorphism (SNP) is located in the CCACC box of the Sp1-binding site, and the T allele is associated with greatly reduced promoter activity [12].

TNF-α is able to induce the expression of MMP-2 in inflamed synovial tissue and blood [13, 14], and has also been implicated in stimulation of the T cell immune response, upregulation of proteolytic enzymes, prostaglandins, and chemokines, and overexpression of adhesion molecules in the pathogenesis of RA. Evidence supports a pivotal role for TNF-α as the prime mediator of inflammation in RA [15]. The TNF-α gene is located on chromosome 6. The TNF-α SNP −308 A/G (rs1800629) in the promoter region has been intensively studied [16], with the A allele being associated with higher levels of TNF transcription [17, 18].

The human NLR family pyrin domain-containing 1 (NLRP1) inflammasome was the first caspase-1-activating protein complex to be identified [19]. NLRP1 interacts with caspase-5, as well as caspase-1, contributing to IL-1β processing in human cells [19]. The NLRP1 gene maps to chromosome 17p13 and has been linked to vitiligo and associated autoimmunity [20]. NLRP1 has been found to confer a risk for extended autoimmune/inflammatory disorders and more recently, for autoimmune Addison’s disease, type I diabetes and RA [2124].

MMP-2, TNF-α and NLRP1 play important roles in the immune response. We therefore performed a hospital-based case–control study with the aim of genotyping MMP-2 rs243865 C/T, TNF-α rs1800629 A/G, NLRP1 rs878329 C/G and NLRP1 rs6502867 C/T polymorphisms in Chinese patients with RA and AS.

Patients and methods

Ethical approval of the study protocol

We obtained approval of the study protocol from the Ethics Committee of Nanjing Medical University (Nanjing, China). All patients provided written informed consent to be included in the study.

Study subjects

Five hundred and twenty RA patients who fulfilled the criteria for RA set by the American College of Rheumatology classification in 1987 [25] were consecutively recruited from the Changzhou Second Hospital-Affiliated Hospital of Nanjing Medical University (Changzhou, China), the Changzhou First Hospital (Changzhou, China), and the Changzhou Traditional Chinese Medical Hospital (Changzhou, China), between September 2010 and May 2012. One hundred AS patients were also recruited from the same three hospitals between March 2011 and May 2012. A diagnosis of AS was established by using the classification criteria reported by the American College of Rheumatology (Modified New York Criteria) [26]. The controls were patients without RA and AS, matched RA for age (±5 years) and sex, and recruited from the same institutions during the same time period; most of the controls were admitted to the hospitals for the treatment of trauma.

Each patient was interviewed by trained personnel using a pre-tested questionnaire to obtain information on demographic data and related risk factors for RA and AS. After the interview, 2 mL of peripheral blood was collected from each subject.

Isolation of DNA and genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Blood samples were collected using vacutainers and transferred to test tubes containing ethylenediamine tetra-acetic acid (EDTA). Genomic DNA was isolated from whole blood using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). Genotyping was done by MALDI-TOF MS using the MassARRAY system (Sequenom, San Diego, CA, USA) as previously described [27]. Cases and controls at a proportion of ≈1:1 were assayed. Completed genotyping reactions were spotted onto a 384-well spectroCHIP (Sequenom) using a MassARRAY Nanodispenser (Sequenom), and analyzed by MALDI-TOF–MS. Genotype calling was done in real time with MassARRAY RT software (version 3.1; Sequenom), and analyzed using MassARRAY Typer software (version 4.0; Sequenom). For quality control, repeated analyses were undertaken on 10 % of randomly selected samples.

Statistical analysis

Differences in demographics, variables, and genotypes of the MMP-2 rs243865 C/T, TNF-α rs1800629 A/G, NLRP1 rs878329 C/G and NLRP1 rs6502867 C/T polymorphism variants were evaluated using a Chi squared test. The associations between MMP-2 rs243865 C/T, TNF-α rs1800629 A/G, NLRP1 rs878329 C/G and NLRP1 rs6502867 C/T genotypes and risk of RA and AS were estimated by computing odds ratios (ORs) and 95 % confidence intervals (CIs) using logistic regression analyses, and by using crude ORs. The Hardy–Weinberg equilibrium (HWE) was tested by a goodness-of-fit Chi squared test to compare the observed genotype frequencies to the expected frequencies among controls. All statistical analyses were done with SAS software (version 9.1.3; SAS Institute, Cary, NC, USA).

Results

Characteristics of the study population

Among 1,140 DNA samples (520 RA patients, 100 AS patients and 520 controls), we successfully genotyped 1,118 (98.1 %) samples in the first step, then in the second step we genotyping the rest 22 samples and 114 (10 % of all samples) randomly selected samples. At last, the MMP-2 rs243865 C/T polymorphism was successful in 520 (100.0 %) RA patients, 100 (100.0 %) AS patients and 520 (100.0 %) controls. Genotyping was successful in 519 (99.8 %) RA patients, 100 (100.0 %) AS patients and 520 (100.0 %) controls for TNF-α rs1800629 A/G, NLRP1 rs878329 C/G and NLRP1 rs6502867 C/T polymorphisms. The concordance rates of repeated analyses were 100 %. The demographic and clinical characteristics of all subjects are summarized in Table 1. Subjects were adequately matched for age and sex (P = 0.496 and 0.722, respectively) for RA patients and controls. Subjects were not adequately matched for age and sex for AS patients and controls (Table 1). The genotype distributions of MMP-2 rs243865 C/T, TNF-α rs1800629 A/G, NLRP1 rs878329 C/G and NLRP1 rs6502867 C/T in all subjects are illustrated in Table 2. The observed genotype frequencies for the polymorphism in controls were in HWE for MMP-2 rs243865 C/T (P = 0.885), TNF-α rs1800629 A/G (P = 0.497), NLRP1 rs878329 C/G (P = 0.065) and NLRP1 rs6502867 C/T (P = 0.950).
Table 1

Patient demographics and risk factors in rheumatoid arthritis and ankylosing spondylitis, all subjects

Variable

Controls (n = 520)

RA cases (n = 520)

P

AS cases (n = 100)

P

Age (years)

54.17 (±10.50)

54.72 (±15.27)

0.496

31.13 (±7.74)

<0.050

Female/male

385/135

390/130

0.722

30/70

<0.050

Age at onset, years, mean ± SD

46.43 (±13.28)

25.47 (±5.49)

Disease duration, years, mean ± SD

8.34 (±9.40)

5.68 (±4.29)

Treatment duration, years, mean ± SD

7.03 (±7.93)

4.82 (±3.72)

RF-positive, No. (%)

414 (79.6 %)

ACPA-positive, No. (%)

269 (51.7 %)

CRP-positive, No. (%)

220 (42.3 %)

39 (39.0 %)

ESR, mm/h

36.93 (±29.41)

DAS28

4.55 (±1.53)

HLA-B27

152.2 (±22.03)

HLA-B27-positive, No. (%)

79 (79.0 %)

Functional class, No. (%)

 I

68 (13.1 %)

0 (0 %)

 II

236 (45.4 %)

73 (73.0 %)

 III

183 (35.2 %)

17 (17.0 %)

 IV

33 (6.3 %)

10 (10.0 %)

RF rheumatoid factor, ACPA anti-cyclic citrullinated peptide antibody, CRP C-reactive protein, ESR erythrocyte sedimentation rate, DAS28 RA disease activity score

Table 2

Logistic regression analysis of associations between MMP-2 rs243865 C/T, TNF-α rs1800629 A/G, NLRP1 rs878329 C/G and NLRP1 rs6502867 C/T polymorphisms and risk of rheumatoid arthritis and ankylosing spondylitis

Genotype

Controls (n = 520)

RA (n = 520)

AS (n = 100)

No. (%)

No. (%)

OR (95 % CI)

P

No. (%)

OR (95 % CI)

P

MMP-2 rs243865 C/T

 CC

420 (80.8)

410 (78.8)

1.00

 

91 (91.0)

1.00

 

 CT

95 (18.3)

102 (19.6)

1.10 (0.81–1.50)

0.548

8 (8.0)

0.39 (0.18–0.83)

0.014

 TT

5 (1.0)

8 (1.5)

1.64 (0.53–5.05)

0.390

1 (1.0)

0.92 (0.11–8.00)

0.942

 CT + TT

100 (19.2)

110 (21.2)

1.13 (0.83–1.53)

0.440

9 (9.0)

0.42 (0.20–0.85)

0.017

 CC + CT

515 (99.0)

512 (98.5)

1.00

 

99 (99.0)

1.00

 

 TT

5 (1.0)

8 (1.5)

1.61 (0.52–4.95)

0.407

1 (1.0)

1.04 (0.12–9.00)

0.971

 T allele

105 (10.1)

118 (11.3)

  

10 (5.0)

  

TNF-α rs1800629 A/G

 GG

446 (85.8)

456 (87.9)

1.00

 

92 (92.0)

1.00

 

 GA

70 (13.5)

63 (12.1)

0.88 (0.61–1.27)

0.493

8 (8.0)

0.55 (0.26–1.19)

0.130

 AA

4 (0.8)

0 (0.0)

0.981

0 (0.0)

0.988

 GA + AA

74 (14.2)

63 (12.1)

0.83 (0.58–1.19)

0.320

8 (8.0)

0.52 (0.24–1.12)

0.097

 GG + GA

516 (99.2)

519 (100.0)

1.00

 

100 (100.0)

1.00

 

 AA

4 (0.8)

0 (0.0)

0.981

0 (0.0)

0.988

 A allele

78 (7.5)

63 (6.1)

  

8 (4.0)

  

NLRP1 rs878329 C/G

 GG

347 (66.7)

334 (64.4)

1.00

 

65 (65.0)

1.00

 

 GC

163 (31.3)

169 (32.6)

1.08 (0.83–1.40)

0.579

32 (32.0)

1.05 (0.66–1.66)

0.842

 CC

10 (1.9)

16 (3.1)

1.66 (0.74–3.72)

0.216

3 (3.0)

1.60 (0.43–5.98)

0.483

 GC + CC

173 (33.3)

185 (35.6)

1.11 (0.86–1.44)

0.421

35 (35.0)

1.08 (0.69–1.69)

0.737

 GG + GC

510 (98.1)

503 (96.9)

1.00

 

97 (97.0)

1.00

 

 CC

10 (1.9)

16 (3.1)

1.62 (0.73–3.61)

0.236

3 (3.0)

1.58 (0.43–5.84)

0.495

 C allele

183 (17.6)

201 (19.4)

  

38 (19.0)

  

NLRP1 rs6502867 C/T

 TT

474 (91.2)

481 (92.7)

1.00

 

94 (94.0)

1.00

 

 TC

45 (8.7)

38 (7.3)

0.83 (0.53–1.31)

0.424

6 (6.0)

0.67 (0.28–1.62)

0.377

 CC

1 (0.2)

0 (0.0)

0.978

0 (0.0)

0.986

 TC + CC

46 (8.8)

38 (7.3)

0.82 (0.52–1.28)

0.370

6 (6.0)

0.66 (0.27–1.58)

0.350

 TT + TC

519 (99.8)

519 (100.0)

1.00

 

100 (100.0)

1.00

 

 CC

1 (0.2)

0 (0.0)

0.978

0 (0.0)

0.986

 C allele

47 (4.5)

38 (3.7)

  

6 (3.0)

  

Bold values are statistically significant (P < 0.05)

Associations between MMP-2 rs243865 C/T, TNF-α rs1800629 A/G, NLRP1 rs878329 C/G and NLRP1 rs6502867 C/T polymorphisms and the risk of RA.

None of the MMP-2 rs243865 C/T, TNF-α rs1800629 A/G, NLRP1 rs878329 C/G and NLRP1 rs6502867 C/T polymorphisms achieved a significant difference in the genotype distributions between cases and controls. Logistic regression analyses revealed that MMP-2 rs243865 C/T, TNF-α rs1800629 A/G, NLRP1 rs878329 C/G and NLRP1 rs6502867 C/T polymorphisms were not associated with the risk of RA (Table 2).

Associations between MMP-2 rs243865 C/T, TNF-α rs1800629 A/G, NLRP1 rs878329 C/G and NLRP1 rs6502867 C/T polymorphisms and the risk of AS.

The genotype frequencies of the MMP-2 rs243865 C/T polymorphism were 91.0 % (CC), 8.0 % (CT) and 1.0 % (TT) in AS patients, and 80.8 % (CC), 18.3 % (CT) and 1.0 % (TT) in controls (P = 0.041) (Table 2). When the MMP-2 rs243865 CC homozygote genotype was used as the reference group, the CT or TT/CT genotypes were associated with a significantly decreased risk for AS (CT vs. CC, OR = 0.39, 95 % CI = 0.18–0.83, P = 0.014; TT/CT vs. CC, OR = 0.42, 95 % CI = 0.20–0.85, P = 0.017). When the MMP-2 rs243865 CC homozygote genotype was used as the reference group, the TT genotype was not associated with the risk for AS (TT vs. CC, OR = 0.92, 95 % CI = 0.11–8.00, P = 0.942). In the recessive model, when the MMP-2 rs243865 CC/CT genotypes were used as the reference group, the TT homozygote genotype was not associated with susceptibility to AS (OR = 1.04, 95 % CI = 0.12–9.00, P = 0.971).

None of the TNF-α rs1800629 A/G, NLRP1 rs878329 C/G and NLRP1 rs6502867 C/T polymorphisms achieved a significant difference in the genotype distributions between cases and controls. Logistic regression analyses revealed that TNF-α rs1800629 A/G, NLRP1 rs878329 C/G and NLRP1 rs6502867 C/T polymorphisms were not associated with the risk of AS (Table 2).

Discussion

We investigated the associations between the MMP-2 rs243865 C/T, TNF-α rs1800629 A/G, NLRP1 rs878329 C/G and NLRP1 rs6502867 C/T polymorphisms and the risks of RA and AS in a Chinese population. The results suggested that the MMP-2 rs243865 C/T polymorphism may be associated with risk of AS. To the best of our knowledge, this is the first demonstration of an association between the MMP-2 rs243865 C/T polymorphism and AS.

Type V collagen is emerging as a central molecule in fibril formation and assembly of the collagen network, and AS patients have been shown to have elevated levels of MMP-2/9-mediated type V collagen degradation [28].

MMP-2 levels are significantly increased in autoimmune diseases with varying degrees of disruption of the immune response. MMP-2 affects the clearance of recruited immune cells, which is necessary for resolving inflammatory reactions [10]. The MMP-2 −1306 C/T (rs243865) polymorphism is located in the CCACC box of the Sp1-binding site, and the T allele is associated with greatly reduced promoter activity [12, 29]. It is likely that the CC genotype is associated with high transcription levels and enzyme activity of MMP-2, while the −1306 T allele produces lower MMP-2 protein levels in individuals carrying the CT or TT genotype. The presence of the T allele at the −1306 C>T polymorphism in the promoter of the MMP-2 gene was also associated with significant and independent protection against abdominal aortic aneurysm [30], while a meta-analysis suggested that the MMP-2 −1306 C>T T allele was associated with decreased risks of lung, head and neck, gastric and esophageal cancers [31]. The T allele was associated with lower MMP-2 expression, followed by reduced cell migration and invasion, which may partly explain its protective effects in AS. Rodriguez-Lopez et al. found that the T allele of the MMP-2 −1306 C>T polymorphism increased the risk of RA in a Spanish population [32]. However, this association was not confirmed in our RA patients, in accordance with the results of a study of RA in a French Caucasian population [33].

The association between the TNF-α rs1800629 A/G polymorphism and RA susceptibility has been controversial [3437], and the current study found no association between this polymorphism and the risks of RA and AS.

Previous studies have investigated the NLRP1 rs878329 C/G and rs6502867 C/T promoter polymorphisms. A study in Han Chinese revealed an association between NLRP1 and RA, indicating that the rs878329 G allele in the NLRP1 promoter up-regulated gene transcription and conferred a risk of RA [24]; however, this association was not confirmed in our study of the rs878329 C/G and rs6502867 C/T polymorphisms.

Considering the MMP-2 rs243865 C/T mutant alleles, the power of our analysis (α = 0.05) was 0.405 for 100 AS cases and 520 controls with an OR of 0.42, and 0.093 for 520 RA cases and 520 controls with an OR of 1.13 for MMP-2 rs243865 C/T.

There were several limitations to the present study. First, because this was a hospital-based case–control study, the subjects were not fully representative of the general population, and selection bias was therefore unavoidable. Second, the polymorphisms investigated may not offer a comprehensive view of the genetic variability of MMP-2, TNF-α and NLRP1, based on their functional considerations. Third, a single case–control study is not sufficient to allow a full interpretation of the relationships between MMP-2 rs243865 C/T, TNF-α rs1800629 A/G, NLRP1 rs878329 C/G and NLRP1 rs6502867 C/T polymorphisms and susceptibility to RA and AS, because of the relatively small number of patients evaluated. Further studies with more subjects are needed to confirm our findings. Finally, environmental factors, as well as genetic factors, differ between Chinese and other populations; because the risks of RA and AS are likely to be influenced by gene–gene and gene-environment interactions, the MMP-2, TNF-α and NLRP1 genes may be associated with different degrees of genetic risk in different ethnic groups and under different environmental conditions.

In conclusion, the present study provided strong evidence to suggest that the MMP-2 rs243865 C/T polymorphism may be associated with reduced risk of AS. Further studies are needed to confirm the results of this preliminary study.

Acknowledgments

This study was supported in part by National Natural Science Foundation of China (81371927) and Nanjing Medical University Foundation for Development of Science and Technology (06NMUZ045, 2012NJMU128).

Conflict of interest

None of the authors has any potential financial conflict of interest related to this manuscript.

Copyright information

© Springer Science+Business Media Dordrecht 2013