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Gene polymorphisms of the MMP1, MMP9, MMP12, IL-1β and TIMP1 and the risk of primary open-angle glaucoma

2013; Wiley; Volume: 91; Issue: 7 Linguagem: Inglês

10.1111/aos.12149

1755-3768

Łukasz Markiewicz, Ireneusz Majsterek, Karolina Przybyłowska, Łukasz Dziki, Maja Waszczyk, Mira Gacek, Anna Kamińska, Jerzy Szaflik, Jacek P. Szaflik,

Retinal Diseases and Treatments

Background: Primary open-angle glaucoma (POAG) is the main cause of irreversible blindness worldwide. Matrix metalloproteinases (MMPs) and their regulators (TIMPs and ILs) have been extensively studied as POAG risk factors. Recent reports have showed several single-nucleotide polymorphisms (SNPs) for MMPs, TIMPs and ILs encoding genes in patients with POAG. The aim of this study was to investigate association of the -1607 1G/2G MMP1, -the 1562 C/T MMP9, the -82 A/G MMP12, the -511 C/T IL-1β and the 372 T/C TIMP1 gene polymorphisms with POAG occurrence and to investigate their impact on main clinical features. Material and methods: In the present case–control study, we examined group of 511 unrelated Caucasian subjects consist of 255 patients with POAG (mean age 70 ± 15) and 256 controls (mean age 67 ± 16). Determination of genes polymorphic variants was made using polymerase chain reaction–restriction fragment length polymorphism technique (PCR-RFLP). The odds ratios (ORs) and 95% confidence intervals (CIs) for each genotype and allele were calculated. Results: Presented study showed statistically significant increase in the POAG development risk of the -1607 2G/2G MMP1 genotype (OR 1.75; 95% CI, 1.11–2.75; p = 0.014) and for the -1607 2G MMP1 allele (OR 1.35; 95% CI, 1.05–1.73; p = 0.017), as well as for the -1562 C/T MMP9 genotype (OR 1.74; 95% CI, 1.17–2.59; p = 0.006) and the -1562 T MMP9 allele (OR 1.55; 95% CI, 1.10–2.17; p = 0.012) in patients with POAG in comparison with healthy control group. We also observed positive association of the -511 T/T IL-1β genotype (OR 2.60; 95% CI, 1.41–4.80; p = 0.002) as well as the -511 T IL-1β allele occurrence with an increased POAG development risk (OR 1.47; 95% CI, 1.13–1.90; p = 0.003). Furthermore, we found an association of the -1607 1G/2G MMP1, -1562 C/T MMP9 (anova, p < 0.001) and the -511 C/T IL-1β gene polymorphism (anova, p < 0.05) with decreased retinal nerve fibre layer (RNFL) thickness in patients with POAG group. Results displayed also an association of the 372 T/C TIMP1 gene polymorphism with normal range RNFL (anova, p < 0.001). We observed an association of decreased RA value (rim area) with the -82 A/G MMP12 (anova, p < 0.001). Normal RA value was observed in patients with POAG group connected with the 372 T/C TIMP1 (anova, p < 0.05) and the -511 C/T IL-1β (anova, p < 0.05) genes polymorphisms occurrence. Finally, results showed an association of the -1562 C/T MMP9 (anova, p < 0.001) gene polymorphism with decreased cup/disc index in patients with POAG group. Conclusion: In conclusion, we suggest that the -1607 1G/2G MMP1, -1562 C/T MMP9, -511 C/T IL-1β gene polymorphisms can be considered as an important risk factors associated with POAG. Primary open-angle glaucoma (POAG) is one of main causes of irreversible blindness worldwide (Resnikoff et al. 2004). This type of eye disease leads to progressive and irreversible damage to the optic nerve and retinal ganglion cells (RGC), and hence the deterioration and eventually loss of vision (Kwon et al. 2009). Recently, epidemiological studies indicated that apart from elevated intraocular pressure (IOP) and age also factors like ethnic origin, diabetes mellitus and most of all genetic predisposition might be associated with a risk of POAG (Flammer et al. 2002; Marcic et al. 2003; Waliszek-Iwanicka et al. 2010). Mutations in the myocilin and optineurin gene (MYOC and OPTN) were considered as the most prominent genetic factors (Cheng et al. 2010; Stone et al. 1997), however, were not found in most of patients with POAG (Alward et al. 2002). Recently, several single-nucleotide polymorphisms (SNPs) in metalloproteinases (MMPs), tissue inhibitors for metalloproteinases (TIMPs) and interleukins (ILs) encoding genes have been identified in patients with POAG (Fujimoto et al. 2002; Krex et al. 2003; Rutter et al. 1998). The matrix metalloproteinases constitute a group of neutral, Ca- and Zn-activated endoproteinases involved mainly in physiological extracellular matrix (ECM) turnover during embryogenesis and angiogenesis but also in interactions between cells and their surrounding (Stone et al. 1997; Visse & Nagase 2003). In the context of POAG, metalloproteinases are responsible for the IOP regulation by affecting aqueous humour outflow from anterior chamber of the eye via the irido-corneal drainage angle (Maatta et al. 2005, 2006). A significantly increased level of MMPs in the aqueous humour of patients with diagnosed POAG has been previously described (Maatta et al. 2005; Schlotzer-Schrehardt et al. 2003). To estimate an association of MMPs and their regulators with POAG, we investigated the -1607 1G/2G MMP1, -1562 C/T MMP9, -82 A/G MMP12, -511 C/T IL-1β and 372 T/C TIMP1 gene polymorphisms in patients with POAG group compared with healthy controls. In the present case–control study, we investigated a total of 511 unrelated Caucasian subjects from Polish population. The study group consists of 255 unrelated patients with diagnosed POAG (mean age 70 ± 15, 90 men and 165 women) and the control group of 256 unrelated patients without glaucoma symptoms (mean age 67 ± 16). All patients and controls were matched on age and sex (no differences were calculated, p > 0.05), controls visual acuity ranged from 20/20 to 20/30. All studied subjects underwent ophthalmic examination, including best-corrected visual acuity, IOP, slit-lamp examination, gonioscopy and fundus examination using noncontact and contact fundus lenses with a slit lamp. In the group of glaucomatous patients, the diagnosis of POAG was stated prior to enrolment in accordance with the guidelines of European Glaucoma Society (Terminology and Guidelines for Glaucoma IInd Edition, Dogma, Savona 2003, Italy). The patients with POAG at the time of enrolling in the study were treated topically with one or the combination of typical antiglaucoma medications including beta blockers (i.e. Timolol), prostaglandin analogues (i.e. Latanoprost) carbonic anhydrase inhibitors (i.e. Dorzolamide) and alpha2 agonists (i.e. Brimonidine). Medical history was obtained from all subjects, and no one reported present or former cancer or any genetic disease. The characteristic of POAG patients group is displayed in Table 1. Patients were excluded from the study if they were subject to any of the following conditions, which could possibly interfere with the results of the study: use of eye drops other than antiglaucoma preparations, any ocular surgeries or laser treatments performed in the past in the eye from which the specimens were to be collected, present or prior treatment with glucocorticosteroids or immunosuppressive therapy (if these treatments had not been stopped at least 1 year before the surgery and collection of specimens), use of nonsteroidal anti-inflammatory drugs (with the exception of low-dose aspirin, which had to be stopped 7 days before the surgery and collection of specimens), prior and concurrent systemic antibiotic treatment during the last 7 days before the start of the study. All subjects included in the study resided in Warsaw District, Poland. All patients were recruited from the Department of Ophthalmology, Medical University of Warsaw. The study was reviewed and approved by the local Ethics Committee (permission no. RNN/468/10/kB) and met the tenets of the Declaration of Helsinki. Written consent was obtained from each patient before enrolment in the study. Blood samples were collected in 3-ml EDTA tubes. DNA was extracted from peripheral lymphocytes using the nucleic isolation kit: QIAamp DNA Mini and Blood Kit (Qiagen, Chatsworth, CA, USA) following the manufactures protocol and stored at −20°C. The -1607 1G/2G MMP1, -1562 C/T MMP9, -82 A/G MMP12, -511 C/T IL-1β and 372 T/C TIMP1 genotypes were determined by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) according to previously described procedures with some modifications (Table 2) (De Souza et al. 2003; Dunleavey et al. 2000; Kubben et al. 2006; Wollmer et al. 2002). Briefly, each 20 μl of the PCR contained 10 ng genomic DNA, 1.25 U Taq polymerase (Qiagen) in 1 × PCR buffer (100 mm Tris-HCl, pH 8.3, 500 mm KCl, 11 mm MgCl2, 0.1% gelatine), 1.5 mm MgCl2, 50 mm dNTPs and 250 nm each primer. Thermal cycling conditions with primer sequences (Sigma-Aldrich, St. Louis, MO, USA) displayed in Table 1. The PCR was carried out in a MJ Research, INC thermal cycler; model PTC-100 (Waltham, MA, USA). Primer sequences used in amplification of the MMP1 -1607 1G/2G and the TIMP1 372 T/C gene polymorphic sites were displayed in Table 2. Two mismatches were introduced into the reverse annealing primer of the MMP1 -1607 1G/2G polymorphism (De Souza et al. 2003) resulting in the restriction endonuclease XmnI recognition sequence (5′-GAANNNNTTC-3′) for the 1G allele. The MMP1 PCR amplification product (118 bp) was digested with 1 unit of XmnI (New England Biolabs, Ipswich, MA, USA) for 16 hr, and only at the presence of the 1G allele, it was cut in two fragments of 89 and 29 bp. The TIMP1 PCR amplification product (175 bp) was digested with 1 units BssSI (New England Biolabs) for 16 hr, and only at the presence of C allele, it was cut in two fragment of 155 and 20 bp. The PCR products were separated by 8% polyacrylamide or 3% agarose gel electrophoresis. The MMP9 PCR amplification product (435 bp) was digested with 1 units SphI (New England Biolabs) for 16 hr, and only at the presence of T allele, it was cut in two fragment of 247 and 188 bp. The MMP12 PCR amplification product (163 bp) was digested with 1 units HpyCH4III (New England Biolabs) for 16 hr, and only at the presence of G allele, it was cut in two fragment of 101 and 62 bp. Finally, the IL-1β PCR amplification product (304 bp) was digested with 1 units AvaI (New England Biolabs) for 16 hr, and only at the presence of C allele, it was cut in two fragment of 190 and 114 bp. The allele frequencies were estimated by gene counting and genotypes were scored. The chi-squared test was used to compare the observed numbers of genotypes with those expected for a population in the Hardy–Weinberg equilibrium (HWE) and to test the significance of the differences of observed alleles and genotypes between groups. A p-value of <0.05 was taken as statistically significant. The odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using reference value (Reference) of wild-type genotype. In addition, when calculating the ORs Pearson's correction was used, and if the expected cell values were less than 5, Fisher's exact test was used. A p-value of <0.05 was taken as statistically significant. The t-test (for normal distribution) or Mann–Whitney U-test (for nonnormal distribution) was used to compare each parameter between two groups. An analysis of variance test was used to identify parameters that would make significant differences between the distributions of more than two genotypes. All positive results of anova test indicating an association of genotypes with clinical parameters of glaucoma were also analysed post hoc with using of Tukey's HSD test (differences between means for genotypes were compared with calculated HSD value). A p-value of 0.05; χ2 = 2.02), MMP9 (p > 0.05; χ2 = 0.172), IL-1β (p > 0.05; χ2 = 2.22) as well as MMP12 (p > 0.05; χ2 = 0.379) in the controls group were in agreement with Hardy–Weinberg equilibrium (HWE). A statistically significant increase in the frequency of the -1607 2G/2G MMP1 genotype (OR 1.75; 95% CI, 1.11–2.75; p = 0.014) and the -1607 2G MMP1 allele (OR 1.33; 95% CI, 1.04–1.71; p = 0.020), as well as the -1562 C/T MMP9 genotype (OR 1.74; 95% CI, 1.17–2.59; p = 0.006) and the -1562 T MMP9 allele (OR 1.55; 95% CI, 1.10–2.17; p = 0.012) in patients with POAG in comparison with healthy controls. We also observed positive association of the -511 T/T IL-1β genotype (OR 2.60; 95% CI, 1.41–4.80; p = 0.002) as well as the -511 T IL-1β allele occurrence with an increased POAG development risk (OR 1.47; 95% CI, 1.13–1.90; p = 0.003). Any association of researched TIMP1 and MMP12 polymorphisms with POAG risk development was found. In addition, the gene–gene co-occurrence of polymorphic variants of studied genes was analysed (Table 4). The assessment showed positive association of the 2G2/G-C/T MMP1-IL-1β genotype (OR 3.16; 95% CI, 1.55–6.47; p < 0.001), the 1G/1G-T/T MMP1-IL-1β genotype (OR 3.56; 95% CI, 1.31–9.63; p = 0.009) and the 2G/2G-T/T MMP1/IL-1β genotype (OR 4.03; 95% CI, 1.17–13.87; p = 0.020) with an increased POAG development risk. Analysis of MMP9-IL-1β genotypes also revealed positive association of the C/T-C/T genotype (OR 2.26; 95% CI, 1.28–3.95; p = 0.004) as well as the C/T-T/T genotype (OR 4.46; 95% CI, 1.39–14.29; p = 0.006) with an elevated risk of POAG development. Finally, analysis showed almost three times higher POAG development risk of the T/T-A/A IL-1β-MMP12 genotype (OR 2.73; 95% CI, 1.30–5.75; p = 0.007) and over four times higher POAG development risk of the T/T-A/G IL-1β-MMP12 genotype (OR 4.27; 95% CI, 1.32–13.83; p = 0.009). Distributions of genotypes and allele frequencies of gene polymorphisms with regard to clinical results are presented in Tables 5–8. Each patient was analysed for the right eye and left eye separately, and the value of clinical parameters were calculated according to the genotypes distribution. We found an association of the -1607 2G/2G genotype of MMP1 gene (anova p < 0.001; RNFL 2G/2G versus 1G/1G ΔΧ>HSD and 2G/2G versus 1G/2G ΔΧ>HSD), the -1562 T/T genotype of MMP9 gene (anova, p < 0.001, RNFL T/T versus C/C ΔΧ>HSD and T/T versus C/T ΔΧ>HSD) with decreased RNFL in patients with POAG group. Results displayed also an association of the 372 C/C genotype of TIMP1 gene with normal RNFL (anova, p < 0.001, RNFL C/C versus T/C ΔΧ>HSD and C/C versus T/T ΔΧ>HSD). We observed an association of decreased RA value (rim area) with the occurrence of the -82 G/G genotype of MMP12 gene (anova, p < 0.001, RA G/G versus A/G ΔΧ>HSD and G/G versus A/A ΔΧ>HSD). Increased RA value was observed in patients with POAG group connected with the 372 C/C TIMP1 genotype (anova, p < 0.05, RA C/C versus T/C ΔΧ>HSD and C/C versus T/T ΔΧ>HSD) and the -511 T/T IL-1β genotype (anova, p < 0.05, RA T/T versus C/C ΔΧ>HSD and T/T versus C/T ΔΧ>HSD). Any statistically significant association of researched genes polymorphisms in POAG patients with nerve fibre index (NFI value) and cup/disc ratio was found. It is estimated that over 68 million people suffer from glaucoma worldwide and there may be more than 750 000 people affected in Poland (Quigley & Broman 2006). First symptoms of POAG are very often difficult to notice, however in some patient's eyes, the resistance to aqueous humour outflow is significantly increased. An elevated plaque-like material and remodelling within the trabecular meshwork (TM) have been linked to the elevated IOP in patients with POAG (Acott & Kelley 2008). The role of matrix metalloproteinases in pathogenesis of POAG is strongly suggested (Flammer et al. 2002; Marcic et al. 2003). Hernandez has identified the astrocytes as the key cell type involved in this process at the optic nerve hypoplasia and has shown astrocytes to be activated by increased IOP (Hernandez 2000; Yan et al. 2000). Yan et al. (2000) have shown that these active astrocytes are responsible for the production of the matrix-degrading enzymes (MMPs) that affect the pattern of matrix remodelling. However, the pathway leading through remodelling of the TM to RGC death is not fully elucidated. MMPs play an essential role in the turnover of the extracellular matrix components, thereby affecting intercellular behaviour (Visse & Nagase 2003). In the present study, the genotype distribution and allele frequencies of the -1607 1G/2G MMP1, -1562 C/T MMP9, -82 A/G MMP12, -511 C/T IL-1β and 372 T/C TIMP1 genes polymorphisms were determined in the group of 255 patients with POAG and 256 control subjects. A statistically significant increase in the -1607 2G/2G MMP1 genotype and -1607 2G MMP1 allele frequency in patients with POAG in comparison with healthy controls were estimated. According to literature data, an additional guanine insertion of 2G allele in the promoter region creates a PEA3 consensus sequence next to the AP-1 binding site resulting in up-regulation of MMP1 gene expression (Fujimoto et al. 2002; Rutter et al. 1998). The signal transduction of in the TM strongly affects MMPs expression. The study conducted by Mookherjee et al. (2010) suggests that the genomic region containing the IL-1 gene cluster influences the POAG pathogenesis. Chua et al. (2012) revealed significant differences in the aqueous cytokine profile of glaucomatous eyes compared with age-matched controls. It was observed that the combinations of IL-1β with TNF-α produced very strong synergistic increases in MMP3 expression (Kelley et al. 2007). Kelley et al. (2007) also showed that the MMP9 response to IL-1β cytokine is also strong resulting metalloproteinase 9 overexpression. IBD patients carrying the T allele at SNP +372 expressed lower levels of TIMP1 compared with those carrying the C allele, (p = 0.009) (Meijer et al. 2007). Previously, a significantly altered level of MMPs and their inhibitors in the aqueous humour obtained from patients with POAG has been reported (Maatta et al. 2005; Schlotzer-Schrehardt et al. 2003). Ronkko et al. (2007) have shown an elevated level of MMPs in the chamber angle of patients with POAG in comparison with normal eyes (Ronkko et al. 2007). Moreover, these results presented an imbalance between total MMPs: MMP1 + MMP2 + MMP3 + MMP9 and their tissue inhibitors: TIMP1 + TIMP-2 + TIMP-3. Furthermore, Ronko's findings indicated a significant excess of MMP1 over its endogenous inhibitor TIMP1 in tissue samples from patients with POAG. An elevated expression of MMP1 in human optic nerve head astrocytes of POAG has been also estimated in work carried out by Agapova et al. (2001) (Agapova et al. 2001). Mossböck et al. (2010) did not observe any correlation of MMP1 genotype with POAG in patients from Austria but a Greek study showed a trend for the -1607 1G/2G polymorphism association with exfoliation glaucoma (Mossbock et al. 2010; Tsironi et al. 2009). An association of the 2G/2G genotype and 2G allele that we found with POAG occurrence in researched group seems to be in agreement with previously presented studies. Our preliminary results obtained earlier for MMPs polymorphisms have been confirmed (Majsterek et al. 2011). In that study, we analysed an association of two polymorphisms of the -1607 1G/2G MMP1 and 372 T/C TIMP1 gene with a risk of POAG occurrence. The data obtained showed statistical significance of MMP1/TIMP1 polymorphisms with the risk of POAG occurrence but clinical parameters were not analysed. In the present extended study of five MMPs polymorphisms based on new selected group of patients, we were able to make large statistical analysis of gene–gene interactions as well as POAG progression according to key clinical parameters including RNFL, RA, NRI and cup/disc ratio. The direct impact of MMP9 expression level on ganglion cell loss was previously described (Chintala et al. 2002). The starting point for that study was an observation that MMP9 was expressed at the low constitutive level in the ganglion cell layer of the retina (Chintala et al. 2002). Guo et al. (2005) found that enhancement of MMP9 activity in apoptotic RGCs paralleled the decreased deposition of laminin in the RGC layer, what suggests the increased degradation of the ECM at the retinal site resulting from IOP exposure. Our data seem to confirm these findings because, we found an association of the -1562 C/T MMP9 genotype with the risk of glaucoma occurrence (OR 1.74) and -1562 T MMP9 allele with the decrease in RNFL (anova p < 0.001; RNFL T/T versus C/C ΔΧ>HSD and T/T versus C/T ΔΧ>HSD). Moreover, according to study of the central nervous system (CNS), it is suggested that rapid induction of pro-inflammatory cytokine IL-1β plays an important role in neuronal degeneration. Zhang & Chintala (2004) suggested that optic nerve ligation-induced IL-1 β promotes retinal damage by increasing MMP9 synthesis in the retina (Zhang & Chintala 2004). Studies performed by Chua et al. (2012) showed that primary glaucoma can be associated with changes in the aqueous cytokine profile. Chua et al. (2012) reported increased levels of IL-9, IL- 10 and IL-12 in glaucomatous patients (Chua et al. 2012). We found that the-511 T allele of interleukin 1β gene may contribute to the increased risk of POAG. Moreover, our results also showed an association of the -1607 2G/2G genotype and the -1607 2G allele of MMP1 with the decrease in RNFL in patients with POAG (anova p < 0.001; RNFL 1G/1G versus 2G/2G ΔΧ>HSD and 1G/2G versus 2G/2G ΔΧ>HSD). It should be noted that both the -511 T IL-1β and the -1607 2G MMP1 alleles are associated with increased gene expression. Furthermore, we observed normal RNFL value associated with the 372 C/C TIMP1 genotype (anova p < 0.001; RNFL C/C versus T/C ΔΧ>HSD and C/C versus T/T ΔΧ>HSD), which is considered as a factor of the low TIMP1 expression (Meijer et al. 2007). It can be assumed that low expression of the 372 C TIMP1 allele causes a similar biological effect as increased MMP1 expression connected with -1607 2G allele of MMP1. Finally, our findings confirm that MMPs expression level can be a crucial risk factor of POAG. The gene–gene interaction of combined genotypes according to their polymorphic variants showed that especially co-occurrence of -511 C/T IL-1β with others may stronger affect a risk of glaucoma occurrence. It was found that the A/G-T/T combined genotype of MMP12 and IL-1β (OR 4.27 versus 1.08 for A/G MMP12 and 2.60 for T/T IL-1β), and the 2G/2G-C/T and 1G/1G-T/T combined genotypes of MMP1 and IL-1β (OR 3.16 versus 1.75 for 2G/2G MMP1 and 1.31 for C/T IL-1β; OR 3.56 versus 0.69 for 1G1G MMP1 and 2.60 for T/T IL-1β) showed higher OR than the individual effect of each genotype evaluated separately. Those results may suggest that the gene–gene interaction may play an important role as an enhancer of POAG risk which needs to be investigated. We did not observe any correlation of the IL-1β polymorphism with RNFL decrease, while Chua et al. (2012) suggested the induction of MMP's expression by interleukins. Our results rather showed the increase in RNFL associated with the -511 T/T genotype of IL-1β. Thus, mechanism of glaucoma development according to the -511 C/T IL-1β polymorphism remains unclear. The data suggest that the -511 C/T IL-1β polymorphism may be considered as a risk factor for POAG occurrence, which is not associated with its progression. Other molecular mechanisms need to be investigated to fully elucidate IL-1β involvement in POAG pathogenesis. We believe that our results may in part, explain an altered TM remodelling in pathogenesis of open-angle glaucoma and role of MMPs and their regulators in pathogenesis of POAG. Moreover, we suggest that understanding of the mechanisms leading to ganglion cell loss in diseases in the optic nerve like POAG is essential for the design of much-needed therapeutic strategies to save sight (Chintala et al. 2002; Wierzbowska et al. 2010). In conclusion, we suggest that the -1607 1G/2G MMP1, -1562 C/T MMP9, -511 C/T IL-1β gene polymorphisms can be considered as an important risk factors associated with primary open-angle glaucoma. This work was supported by grants N N402 591240 and N 402 248936 from Polish Ministry of Science and Higher Education, and Young Scientists Grant no. 502-03/724-04/502-54-019.