Abstract
Aims Multiple sclerosis (MS) is an autoimmune, inflammatory disease characterized by loss of myelin forming oligodendrocytes and changes in the blood–brain barrier. Matrix metalloproteinase (MMP) -2 and -9 are known to cause disruption of the blood–brain barrier, remodeling of the basal lamina, regeneration of axons, and remyelination in MS. The imbalance between MMPs and tissue inhibitor metalloproteinases (TIMPs) may lead to the emergence of pathological processes such as MS. The roles of MMP2-1306 C/T and TIMP2-418 G/C genetic variants in MS have not been studied before. We aimed to investigate whether MMP2-1306C/T and TIMP2-418 G/C gene variants are risk factors for patients with relapsing remitting multiple sclerosis (RRMS).
Methods The study included 102 RRMS and 102 healthy controls. Genomic DNA was extracted from peripheral leukocytes from ethylenediaminetetraacetic acid anticoagulated blood. Genotyping of the MMP2-1306C/T and TIMP2G-418C polymorphisms was performed using real-time PCR.
Results There were significant differences in terms of distribution of genotype (MMP2-1306- CT, TT) and T allele frequency between the patients with RRMS and the control group (p<0.0001; p<0.0001). The groups were not different in terms of TIMP2G-418C polymorphisms.
Conclusions In the RRMS group, the genotype and allele frequencies of MMP2-1306C/T polymorphism showed significant differences from the controls. These results indicate that MMP2 might play a role in the pathogenesis of MS even during the inflammation stage.
Significance of this study
What is already known about this subject?
Matrix metalloproteinase (MMPs) play an important role in initiating and perpetuating the inflammatory process in multiple sclerosis (MS).
The studies have shown the up-regulation of MMP-2, MMP-7, MMP-9, and MMP-12 in the brain of patients with MS.
The studies revealed that common non-human leucocyte antigen alleles (such as IL7RA, IL2RA, CLEC16) exert only modest effects on risk of MS.
What are the new findings?
There were significant differences in terms of distribution of genotype (CT, TT) and T allele frequency between the patients with relapsing remitting multiple sclerosis (RRMS) and the control group in this study.
The MMP2-1306 T allele carriers had a significantly increased risk of RRMS compared with the CC homozygotes (OR 3.58).
RRMS and control groups were not different in terms of TIMP2G-418C polymorphisms.
These results indicate that MMP2 might play a role in the pathogenesis of MS even during the inflammation stage.
How might these results change the focus of research or clinical practice?
Results of this study will shed light on the selection of candidate genes which will be investigated in large patient groups which include primary and secondary progressive MS.
Introduction
Multiple sclerosis (MS) is an autoimmune, inflammatory disease of the central nervous system, characterized by demyelination of white matter, axonal injury, changes in the blood–brain barrier (BBB) and leukocyte infiltration.1 ,2 Matrix metalloproteinases (MMPs) are effectors of crucial pathogenetic steps, such as BBB breakdown, invasion of brain parenchyma by immune cells and demyelination in MS.3–6 Specifically, the role for BBB disruption by MMP-2 and -9 has been documented,5 ,7–9 and many authors have shown the up-regulation of MMP-2, MMP-7, MMP-9, and MMP-12 in the brain of patients with MS.10–12 Levels of MMP-9 are elevated in MS, and are predictors of the occurrence of new active lesions through MRI.5 ,13 The elevated serum MMP-9 levels in MS have also been found to correlate with increased gadolinium enhancements during the month of an examination by MRI or during the following month.5 ,7 ,14 Gelatinases (MMP-2 and MMP-9) are also involved in angiogenesis, neurogenesis, remodeling of the basal lamina, regeneration of axons, remyelination, and apoptosis in MS.8 TIMPs which are specific tissue inhibitor of metalloproteinases, play a key role in controlling MMP activity. TIMP-2 is a major regulator of MMP-2. The imbalance between MMPs and TIMPs may result in the emergence of pathological processes.15
Although the biological significance of MMP2-1306C/T and TIMP2-418 G/C genetic variants is well known in many other diseases, their significance in MS has not been studied before.16–21 We aimed to investigate whether MMP2-1306C/T (rs 243865) and TIMP2-418 G/C (rs 8179090) gene variants are risk factors for RRMS in this study.
Materials and methods
Sample collections
The study was designed as a case–control study. Both patients and controls were collected by the Gaziosmanpasa University Medical Faculty. Patients with any malignity, cardiovascular problems, Diabetes mellitus or autoimmune disease (other than MS), primary and secondary progressive MS, and patients with CIS were excluded. The diagnosis of MS was based on the 2005 Revised McDonald Multiple Sclerosis criteria for classification.22 Clinical data collected included gender, age at disease onset, clinical course, and Expanded Disability Status Score (EDSS). The control group was selected from healthy individuals without MS or other aforementioned diseases (malignity, cardiovascular problems, diabetes mellitus, autoimmune disease) in the same age group. Consequently, the study group consisted of 102 unrelated patients with relapsing remitting multiple sclerosis (RRMS) followed by our neurology clinic, and 102 healthy volunteers living in the same region. All participants, patients and healthy controls, were Caucasian and of Turkish origin from the inner Central Black Sea region of Turkey. The study protocol was approved by Gaziosmanpaşa University Ethical Committee (13-KAEK-200); and written informed consent was obtained from each participant.
DNA extraction and genotyping
The High Pure PCR Template Preparation Kit (Roche Molecular Biochemicals, Manheim, Germany) was used to extract the genomic DNA from peripheral leukocytes from EDTA anticoagulated blood. Using real-time PCR to analyze the two groups DNA, MMP2-1306C/T (rs 243865) and TIMP2-418G/C (rs 8179090) polymorphisms were detected. 5′-ACT TTC TTC TCC AGT GCC-3′/5′-TAA ACT AGT AAA GAC AAT CAA GGA AGG-3′ for the MMP2-1306C/T polymorphism, and 5′-GGG ATC CTG TCA GTT TCT CAA TA-3′/5′-CCC TTC AGC TCG ACT CT-3′ for the TIMP2-418G/C polymorphisms were the primers used. The probes for MMP2-1306C/T were Sensor 5′-ACC CAG CAC TCC ACC TCT TT-Fluo-3′/Anchor5′- LCRed-640-CTC TTC AGG TCT CAG CTC AGA AGT CAC TTC-Pho-3′; for TIMP2G-418C, they were Sensor 5′-CCC CGG GGT CCC TTC GAG-Fluo-3′/Anchor 5′- LCRed-640-CAG CCT CGG GGC GAG-Pho-3′. Synthesitisation were carried out by Metabion International AG (Martinsried, Deutschland) for both the primers and the fluorescent-labeled probes.17 PCR and melting curve analyses were carried out in 20 μl glass capillaries (Hoffmann-La Roche).23 MMP2-1306C/T PCR cycling was performed for 600 s at 95°C for DNA denaturation, 35 cycles (10 s at 95°C (denaturation), 15 s at 52°C (annealing), and 25 s at 72°C (extension)). A melting curve analysis was performed after the PCR, by heating to 95°C for 30 s, 45 s at 60°C, and 1 s at 80°C followed by cooling to 40°C for 30 s. 600 s at 95°C for DNA denaturation, 45 cycles (10 s at 95°C (denaturation), 15 s at 50°C (annealing), 25 s at 72°C (extension)) were TIMP2-418G/C cycling conditions. A melting curve analysis was performed by heating to 95°C for 60 s, 90 s at 40°C, and 1 s at 75°C followed by cooling to 40°C for 30 s. Plotting the first negative derivative of the fluorescence with respect to the temperature allowed us to calculate the corresponding melting peaks.
Statistical analysis
Continuous variables were reported as average (Av) and SD. Categorical data were expressed as counts and percentages. The Kolmogorov-Smirnov normality test was used to determine whether continuous variables showed normal distribution. Two independent sample t tests were used to compare continuous data between two groups. The χ2 test or the Fisher's exact test was used to compare the distribution of MMP2-1306C/T and TIMP2-418G/C polymorphisms of patients with RRMS with that of controls. ORs and 95.0% CIs were calculated whenever the χ2 or Fisher's exact test was significant. The χ2 test was used to evaluate the Hardy-Weinberg equilibrium for the distribution of the genotypes of the patients and the controls. Obtained significant probability values were also corrected for multiple testing (Bonferroni correction; Pc). A p value of less than 0.05 was considered to be statistically significant. Statistical analysis was performed using the Statistical Package Program for the Social Sciences (SPSS 20) and the Open Epi Info software package V.3.01.
Results
The patient group consisted of 76 females and 26 males, and the control group 75 females and 27 males. The mean age was 35.93 years for the control group, and 36.69 years for the patient group. There was no significant difference in the mean age and sex ratio between the two groups (p>0.05) (table 1). RRMS patient's age, sex, the mean EDSS score, duration of the disease, frequency of the relapses, and other demographics were shown in table 2.
The distribution of the genotype and allele frequency of MMP2-1306C/T and TIMP2G-418C in patients with RRMS and controls is shown in table 3. The genotype distribution of polymorphisms did not deviate from the Hardy-Weinberg equilibrium in any group. There were significant differences in terms of distribution of genotype (CT, TT) and T allele frequency between the patients with RRMS and the control group (p<0.0001; p<0.0001, OR: 3.58). These results showed that T allele of C1306T polymorphism was associated with RRMS susceptibility in this population. No significant difference was noted for the TIMP2 418G/C polymorphism between the groups (p>0.05) (table 3).
Discussion
This is the first study investigating MMP2-1306C/T and TIMP2G-418C single nucleotide polymorphism in the patients with RRMS. While we found an association between MMP2-1306C/T polymorphisms and RRMS, no association was identified between TIMP2G-418C and RRMS. Altered MMP regulation is associated with pathological processes including inflammation, cell proliferation, cell death and tissue remodeling.24 Although the TIMPs are recognized inhibitors of the MMPs, several studies have revealed that these proteins also can exhibit biological activities that are distinct from their interactions with or inhibition of the MMPs.25 TIMP-2 mRNA is strongly expressed in healthy neurons in all regions of the CNS. TIMP-2 knockout mice exhibit severe motor defects.6
MMP2 (gelatinase A), among other MMPs, primarily hydrolyzes type 4 collagen the major structural component of basement membrane.26 MMP2 is activated at the cell surface by membrane-bound MMP14 (MT1-MMP). Activation implicates formation of a trimolecular complex of pro-MMP2, TIMP2, and MMP14.27 By binding the complex to regions close to the membrane, MMP14 constrains the action of MMP-2. Bar-Or et al28 showed higher levels of monocyte derived MMP2 and MMP14 transcripts and of TIMP2 in MS samples compared to those from normal individuals. Increased expression of MMP related to the high migratory capacity of monocytes (MMP-2, MMP-14, TIMP-2), signifies its role on the neuroinflammatory process in MS. The tripartite of MMP-2, MMP-14, and TIMP-2 could be targets for therapeutic intervention in MS to alleviate the entry of monocytes into the CNS.28 Furthermore, MMP2, MMP14, and TIMP2 are activators of endothelin 1, which is a strong vasoconstrictor that would further compromise blood flow to the deep white matter. It has also been reported that intrathecal MMP-2 production was increased in relapsing-remitting patients with MS.24
MMP-2 is known to play an important role in transport of active T cells into brain parenchyma.6 ,29 Likewise the treatment with AG-3340, an MMP2 inhibitor for knock-out rats, was reported to reduce the MMP2 damage on the blood–brain barrier and as a result to have much less myelin damage.8 It was said that MMP2, which is required to be active during the repair stage of the BBB damage due to disturbance of MMP9 levels and enables the remodeling of extracellular matrix, might be able to transform the course of the condition to a progressive character.3
In our study group the MMP2-1306T allele carriers had a significantly increased risk of RRMS compared with the CC homozygotes. In a study on polymorphisms of MMP2-1306C/T, Gouda et al30 reported that non-Hodgkin's lymphoma risk for persons with T allele was quadrupled. Studies performed in Northern India showed a correlation between bladder/prostate cancer and T allele and TT genotype.31 ,32 In other studies focusing on MMP2-1306C/T polymorphism, patients with lung cancer and gastric adenocarcinoma were shown to have high level of CC genotype which could be related to neoplasmic tendency of this genotype.26 ,33 ,34 A very recently published study reported that the MMP2-1575 G/A polymorphism was of importance for earlier MS onset among patients with ON as an initial symptom.35 In another study, Benesova and et al stated that carriage of the −1575 G allele may influence the MS disease process through higher MMP-2 serum level production. No significant difference was detected for the MMP2-1306C/T polymorphism between the groups in the same study.36 However, this study was carried out in all MS types. For TIMP-2, Zhou et al34 found a reduced risk of breast cancer for the variant allele (−418 GC or CC), compared with the GG common allele. In other studies, the variant allele has been associated with an increased risk of head and neck cancer,18 oral squamous cell cancer,37 and gastric cancer.38 No statistically significant difference was noted between patients with RRMS and control group in our study in terms of TIMP2 418G/C polymorphism (p>0.05).
The most important limitation of this study is that the numbers of cases and controls were small. The first reason for this is that we wanted to conduct the polymorphism study in a single type of MS (RRMS). Moreover, we excluded the diseases that may related to MMP2 such as cardiovascular diseases, malignancy and other autoimmune diseases. In the patient with RRMS group, where neuroinflammation is known to play significant role, the genotype and allele frequencies of 1306C/T polymorphism showed statistically significant differences from the controls (p<0.0001). These results may suggest that MMP2 plays a major role on MS pathogenesis from the time when inflammation was dominant. Inappropriate expression of MMP2 may contribute to the initial pathogenesis of MS.
MS is characterized by a polygenic heritable component that involves multifaceted interactions with environmental factors.39 Genome-wide association studies (GWAS) have revealed that common non-HLA MS risk alleles (such as IL7RA, IL2RA, CLEC16) exert only modest effects on risk39–41 We have detected strong association of MMP2-1306C/T polymorphism and RRMS. The association we observed could be due to the fact that our analysis was performed in a small sample of a particular ethnic group.
When the database of GWAS was examined, we did not encounter any study conducted with large groups concerning MMP2-1306C/T Single nucleotide polymorphism.39–41 Therefore, results of this study will shed light on the selection of candidate genes which will be investigated in large number of patients with MS. Investigating the MMP2-1306C/T polymorphism in patient groups which include primary and secondary progressive MS might provide important information on etiopathogenesis and genetics of MS.
Footnotes
Contributors DA and ÖA contributed in collecting of the data, DNA extraction and genotyping and writing. SK, BÇ and OS have contributed in statistical analysis, writing and reviewing.
Competing interests None declared.
Patient consent Obtained.
Ethics approval Gaziosmanpaşa University Ethical Committee (13-KAEK-200).
Provenance and peer review Not commissioned; externally peer reviewed.