Matrix metalloproteinase-2 (MMP-2 ) and-9 (MMP-9) gene variants and microvascular complications in type 2 diabetes patients

Globally, diabetes mellitus (DM) is a growing medical and social problem. It is estimated that the number of people affected by diabetes will rise from 463 million in 2019 to 700 million by 2045 [1]. Vascular complications lead to increased morbidity and mortality of these patients. The incidence is growing of diabetic microvascular complications, including diabetic retinopathy (DR), diabetic polyneuropathy (DPN), and diabetic nephropathy (DN) [2, 3, 4]. The pathological mechanisms that are responsible for developing these complications are very complex and not fully understood. It seems that genetic susceptibility plays a role in developing such complications [5]. It has been postulated that zinc-dependent endopeptidases called matrix metalloproteinases (MMPs), are very important in remodelling the extracellular matrix, and therefore they have an impact on the onset and progression of diabetic vascular complications [6, 7]. The regulation of MMPs is very complex, depending not only on the regulation of their tissue inhibitors, but also on the regulation of MMP gene expressions by transcriptional factors and epigenetic modifications [8]. Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) are enzymes which are responsible for the degradation of the basal membrane through collagen IV cleveage and therefore have a role in angiogenesis.

Diabetic retinopathy is a very common microvascular diabetic complication, affecting up to one third of patients with diabetes [9, 10]. It can lead to seriuous vision impairments and vision loss, especially in the adult working population [11]. The duration of diabetes and poor metabolic control, as well as elevated blood pressure can contribute to the development of DR [12, 13, 14]. Genetic factors also seem to have an important role, and they account for 25% to 50 % of the risk of developing DR [15]. The role of MMP-2 and MMP-9 is recognized even in the early phases of DR [16] as well as in the latter stages or in the proliferative retinopathy [17, 18]. In the pathogenesis of DR, activated MMPs cause the damage of mitochondria and augment retinal capillary cell apoptosis [19]. Only a few studies regarding the MMPs gene single nucleotide polymorphism (SNP) studies have been completed, and the results of their correlation with DR are conflicting [20, 21].

Diabetic polyneuropathy (DP) is the most prevalent diabetic complication which affects almost half of the patients with diabetes [22]. It can lead to major disability due to foot amputation, and DP significantly reduces the quality of life [23]. It has been shown that MMPs and their inhibitors may have a role in the process of the demyelization and the regeneration of axons [24, 25]. MMP-2 and MMP-9 play a critical role in the pathogenesis of DP [26].

A single genetic study has shown that SNP -1562C>T in the promoter of the MMP-9 gene is seen in patients with diabetic foot ulcers and that increased MMP-9 production from the high expressing T allele may promote matrix degradation [27].

Diabetic nephropathy (DN) develops in 20-40 % patients with diabetes [28] and is the leading cause of end-stage renal disease [29]. Genetic studies regarding the association of MMP gene polymorphism and DN provide conflicting results. Some show that certain polymorphisms of MMP-9 may be predictive factors in the development of diabetic nephropathy [30] and some suggest the protective role of the T allele SNP - 1562C>T of MMP-9 against diabetic nephropathy [31, 32]. It has also been shown that MMP-2 gene polymorphism is associated with susceptibility and disease progression of DN [33].

The objectives of our study were to assess the frequencies of the MMP-2 gene variant NC_000016.9:g.55511806C>T (-1306C>T) and MMP-9 variant NC_000020.10:g.44635976C>T (-1562C>T) in type 2 diabetes patients and healthy subjects and to study whether there is a possible association with the presence of microvascular complications in type 2 diabetes patients.

Statistical analysis was performed using Statistical Package for Social Sciences 20.0 (SPSS Inc., Chicago, Illinois, USA). Descriptive statistics were computed as mean values and standard deviations for continuous variables, and as absolute frequencies and percentage values for categorical variables.

The association between the MMP-2 (-1306 C>T) and MMP-9 (-1562 C>T) variants with disease incidence was examined according to the dominant genetic model (carriers of wild type genotype vs. heterozygous and homozygous carriers), using Pearson’s chi-squared test (χ2). The measure of the strength of the association between genotypes and disease incidence was expressed as an Odds Ratio with a confidence interval of 95% CI (Confidence Interval). A p-value less than 0.05 was considered statistically significant.

The study population included 102 diabetic patients, 65 men and 37 women, aged 61.4 ± 7.3 years with a mean BMI 28.8±4.8kg m2. The mean duration of diabetes was 10.1±5.8 years. Most of the patients were on metformin (86.3 %), 22.5 % on oral antidiabetic drug (OAD), 13.7 % on insulin and 63.7 % on an OAD/insulin combination. The basic demographic features of patients with diabetes are shown in the Table 1. The control group included 56 patients who did not differ in basic demographic characteristics.

In this study, the MMP-2 variant -1306C>T and the MMP-9 variant -1562C>T were analyzed in diabetic patients and in healthy controls. The distribution of genotypes obtained for both variants in the patients with diabetes and the controls is presented in Table 2.

Based on data given in table 2, the frequencies of the -1306C and -1306T alleles in the group of diabetic patients are 0.82 and 0.18, respectively. In the control group, the frequencies of these alleles are 0.71 for -1306C and 0.29 for -1306T. In the group of diabetic patients, the frequencies of -1562C and -1562T alleles are 0.84 and 0.16, respectively; in the control group their frequencies are 0.86 and 0.14, respectively.

For the analyzed alleles, Hardy-Weinberg equilibrium testing was performed. The testing showed that the p value of Pearson’s Chi-Square test for MMP-2 alleles was 0.71 in the group of patients and 0.58 in the control group. For MMP-9 alleles, the p value of Pearson’s Chi-Square test was 0.48 and 0.21 in the group of diabetic patients and in the control group, respectively.

The association of MMP-2 and MMP-9 alleles with diabetes and microvascular complications was investigated in a dominant model of inheritance.

Genotype frequencies of the MMP-9 variant -1562C>T between the patients and control group were not significantly different, as was neither the difference in the C nor T allelic frequencies between the control group and patients with type 2 DM.

The MMP-2 variant -1306C>T showed a negative correlation with type 2 diabetes (p=0.028). The presence of the -1306C allele increases the probability of developing type 2 diabetes 2.2 times, while the -1306 T allele a has protective role.

In order to assess the impact of both variants on microvascular complications, the alleles’ frequencies were correlated with the presence of diabetic retinopathy, polyneuropathy, and nephropathy in patients with type 2 diabetes. The MMP-9 variant 1562C>T showed no association with any microvascular complications. The MMP-2 variant -1306T showed a negative correlation with diabetic polyneuropathy (p=0.017, Table 3). The presence of the -1306C allele increases the probability of developing diabetic polyneuropathy 3.4 times, while the -1306T allele has protective role.

The MMP-2 variant 1306C>T showed no association with the presence of diabetic retinopathy or diabetic nephropathy.

In our study, our primary goal was to assess the possible differences in MMP-2 and MMP-9 gene polymorphism between patients with type 2 diabetes and healthy controls. Our results showed that the presence of the MMP-2 allele -1306C nearly doubles the risk of developing type 2 diabetes mellitus, while the -1306T allele provides protection against this disease. This correlates with the results of Sarray et al., who, for the first time, showed an association of the MMP-2-1306C>T gene variant with the susceptibility to developing type 2 diabetes, therefore confirming the protective effect of the T allele. [35]

To our knowledge, this is the first study investigating MMP-2 and MMP-9 genetic variants and all three microvascular complications.

Regarding DR and a possible genetic background, Singh K. et al. postulated that the functional SNP -1562C>T in the promoter of the MMP-9 gene plays a very important role in developing proliferative DR due to the elevated MMP-9 production from the high expressing T allele leading to retinal angiogenesis [20].

A statistically significant difference was observed in both allele and genotype distributions, but only between patients with proliferative retinopathy versus healthy controls with no DM. There were, however, no significant differences between patients with DM, no matter if they have DR or not. This is the same result as our study. Due to the limited number of patients with proliferative retinopathy we were not able to establish if there is a significant difference between these patients and healthy controls.

In another study, Beranek M. et al. showed that plasma levels of MMP-2 were significantly higher in patients with proliferative DR who were carriers of the -1306 CC or the -1306 CT genotypes [21]. In Beranek’s study, no major differences were found among the groups (diabetics with non-proliferative DR, diabetics with proliferative DR, and healthy controls) when comparing the genotype distribution for MMP-2-1306C>T and MMP-9-1562 C>T variants. This is consistent with our results.

MMPs are regarded as very important players in the development of diabetic polyneuropathy, not only by causing extracellular matrix abnormalities but also by causing neuronal injury which leads to the development of neuropathic pain. So far, no genetic study has been performed regarding MMP gene variants and the presence of DPN. Our results showed that the presence of the MMP-2 allele -1306C increases the probability of developing diabetic polyneuropathy by a factor of 3.4 times. This can be potentially used as genetic marker for detecting those diabetic patients who are at risk of developing this threatening vascular complication. Of course, we need more extensive studies to confirm this finding.

We found no correlation between either MMP-2 or MMP-9 gene variants and diabetic nephropathy. This was probably due to the limited number of patients with this complication in the study group.

In conclusion, our study showed that the variant -1306C>T in the MMP-2 gene doubles the risk of developing type 2 diabetes, and we showed, for the first time, an association of this gene variant and the presence of diabetic polyneuropathy. As matrix metalloproteinases play an important role in the development of vascular diabetic complications, future studies with a larger number of patients are needed so as to verify variants in the genes for MMPs as markers for diabetic complications.