Michal Beránek

Matrix metalloproteinase-9 and matrix metalloproteinase-2 as biomarkers of various courses in multiple sclerosis.

Background Matrix metalloproteinases are notable contributors to neuroinflammation and blood-brain barrier disruption in multiple sclerosis (MS). Objective The goal of this study was to determine the serum levels of matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-2 (MMP-2), and their tissue inhibitors (TIMP-1) and (TIMP-2), and to investigate their possible relations to type, disability, and severity of MS. Materials and methods Eighty-seven patients with definite MS according to the McDonald criteria and 50 healthy controls were enrolled in the study. Their clinical status was evaluated with the Expanded Disability Status Scale. Serum levels were analyzed by enzyme-linked immunoassay. Results A significant elevation in MMP-9 serum levels and in the MMP-9/TIMP-1 ratio was found in the whole MS group (P < 0.001), in the relapsing–remitting MS (RRMS) (P < 0.001), and secondary-progressive MS (SPMS) (P < 0.001) groups when compared with the controls. A significant elevation in MMP-2 serum levels and in the MMP-2/TIMP-2 ratio was observed in the primary progressive (P < 0.001) and the SPMS (P < 0.002) groups when compared with the RRMS group, and this increase was also associated with the disability (P < 0.001) and severity (P < 0.05) of the disease. Conclusion We confirmed that metalloproteinases are useful biological markers in MS, providing information about the clinical type, disability, and severity of the disease.

Polymorphisms in the RAGE gene influence susceptibility to diabetes-associated microvascular dermatoses in NIDDM

To examine genetic polymorphism in the complete sequence of the Receptor of Advanced Glycation End products (RAGE) gene and its possible associations with diabetes-associated microvascular dermatoses (DAMD). Further, to analyze the distribution of individual genotype combinations on the particular polymorphic loci in the RAGE gene. A part of the RAGE gene spanning a region from -4 to 3334 bp was analyzed on a set of 45 subjects with non-insulin dependent diabetes mellitus (NIDDM) and parallel DAMD by means of PCR with subsequent heteroduplex and single-strand conformation polymorphism (SSCP) analyses. Allele frequencies and genotype combinations of novel common polymorphisms were determined in an associations study comprising four groups of subjects (n=390). Fourteen novel polymorphisms (R77C, V89V, 718G/T, 1704G/T, 1727A1728ins, H305Q, S307C, 2117A/G, 2184A/G, 2245G/A, 2249A/G, 2741G/A, and 3089ACdel) and one described previously (G82S) were identified. Significant association with microvascular dermatoses (MD) irrespective of NIDDM were found for exon mutation 82S (P= .004, after a correction for the number of comparisons P(corr) < .05) and marginally significant for intron variant 1704T (P= .032, P(corr)> .05). Calculated odds ratios for 82S and 1704T were 4.73 (95% CI, 1.51 to 14.77) and 1.73 (95% CI, 0.93 to 3.22), respectively. Certain individual genotype combinations of G82S, 1704G/T, and 2184A/G were significantly associated with the presence of MD (P= .00647) both in diabetic and non-diabetic study populations. The two novel polymorphisms (1704G/T and 2184A/G) together with the G82S were shown to influence the susceptibility to MD independent of diabetes itself.

Genetic risk factors for diabetic nephropathy on chromosomes 6p and 7q identified by the set-association approach.

Aims/hypothesisIn the present study we investigated potential associations of a set of 45 single nucleotide polymorphisms (SNP) in 20 candidate genes on eight chromosomes with diabetic nephropathy (DN) in type 2 diabetes mellitus. We aimed to compare two methodological approaches suitable for analysing susceptibility to complex traits: single- and multi-locus analyses.Materials and methodsThe study comprised a total of 647 subjects in one of three groups: diabetes with or without DN, or no diabetes. Genotypes were detected by PCR-based methodology (PCR only, PCR plus RFLP, or allele-specific PCR). Haplotypes were inferred in silico. Set association (tested using SUMSTAT software) was used for multilocus analysis.ResultsAfter correction for multiple comparisons, only one SNP, in the gene encoding the receptor of advanced glycation end products, AGER 2184A/G (gene symbol formerly known as RAGE) showed a significant association with DN (p = 0.0006) in single-locus analysis. In multi-locus analysis, six SNPs exhibited a significant association with DN: four SNPs on chromosome 6p (AGER 2184A/G, LTA 252A/G, EDN1 8002G/A and AGER -429T/C) and two SNPs on chromosome 7q (NOS3 774C/T and NOS3 E298D), omnibus p = 0.033. Haplotype analysis revealed significant differences between DN and control groups in haplotype frequencies on chromosome 6 (p = 0.0002); however, there were no significant difference in the frequencies of the NOS3 haplotypes on chromosome 7. Logistic regression analysis identified SNPs AGER 2184A/G and NOS3 774C/T, together with diabetes duration and HbA1c, as significant predictors of DN. Testing for interactions between SNPs on chromosomes 6 and 7 did not provide significant evidence for epistatic interaction.Conclusions/interpretationUsing the set-association approach we identified significant associations of several SNPs on chromosomes 6 and 7 with DN. The single- and multi-locus analyses represent complementary methods.

Matrix metalloproteinase-9 and matrix metalloproteinase-2 gene polymorphisms in multiple sclerosis

We investigated the association of matrix metalloproteinase-9 (-1562C/T, +279R/Q) and matrix metalloproteinase-2 (-1575G/A, -1306C/T) gene polymorphisms with multiple sclerosis (MS) susceptibility, gender differences and disability in 244 patients and 132 healthy subjects. A significant decrease of the -1562T allele carriers in MS patients compared to controls (Pa=0.01, Pacorr=0.05) in -1562C/T MMP-9 gene polymorphism was found, (odds ratio (OR) -0.58, 95% confidence interval (CI):0.38-0.89). Significant differences were also demonstrated between female patients and healthy females (Pa=0.01, Pacorr=0.05), (OR-0.53, 95% CI:0.32-0.86). Other polymorphisms were not associated either with MS susceptibility or with phenotype of the disease. No association with disability was found.

Duration of Non-Insulin-Dependent Diabetes mellitus and the TNF-β NcoI Genotype as Predictive Factors in Proliferative Diabetic Retinopathy

The object of the study was to investigate the share of the polymorphisms I/D ACE, endothelin 1 4127G/A and TNF-β NcoI in the susceptibility to proliferative diabetic retinopathy (PDR) in non-insulin-dependent diabetes mellitus (NIDDM). Genotypes were detected by polymerase chain reactions and determined in a set of 246 Caucasian NIDDM subjects with defined PDR status. The relevance of genotypes and clinical characteristics to the PDR occurrence was tested using multiple linear regression models and discrimination analysis. The best predictive value for PDR was given by a combination of two parameters – NIDDM duration and the TNF-β genotype (p < 1·10–6 and p = 1·10–2, respectively) with a correct retrograde prediction of 82.6%. A comparison of the TNF-β NcoI allele frequencies revealed no difference between NIDDM and nondiabetic subjects (n = 176), but a statistically significant difference was found between PDR and non-PDR NIDDM subjects (after a correction for the number of comparisons p = 0.03), allele β2 being associated with PDR. Our results identified the allele variant TNF-β2 being associated with PDR in NIDDM. Diabetes duration and the TNF-β NcoI genotype were proven to significantly predict PDR occurrence. The TNF-β2 allele could be regarded as a separate genetic risk factor that increases the relative incidence of PDR in patients with NIDDM.

Polymorphisms 1704G/T, 2184A/G and 2245G/A in the RAGE gene are not associated with diabetic retinopathy in NIDDM - Pilot study

Pilot study perfromed on 531 Caucasian subjects proved that intron polymorphisms 1704G/T, 2184A/G and 2245G/A in the gene encoding receptor of advanced glycation end products (RAGE) are not associated with proliferative diabetic retinopathy in NIDDM.

Polymorphisms in the von Willebrand factor gene are not associated with proliferative retinopathy in non-insulin-dependent diabetes mellitus.

Von Willebrand factor, a multimeric glycoprotein synthesized mainly by endothelial cells, is involved in platelet adhesion and aggregation and has an important role in the process of angiogenesis. Increased levels of von Willebrand factor, reflecting activation or damage of endothelial cells, have been described in association with proliferative diabetic retinopathy (PDR). We investigated the relationships of two polymorphisms (–1793G/C and Thr789Ala) in the von Willebrand factor gene with PDR. Genotypes were detected by polymerase chain reactions with subsequent restrictions with specific endonucleases. Allele frequencies were determined in an association study (n = 371) comprising three groups of subjects (diabetics with and without retinopathy and nondiabetics). Allele frequencies of the –1793G/C and Thr789Ala did not differ between the non-insulin-dependent diabetes mellitus (NIDDM) + PDR and the NIDDM non-PDR groups (p > 0.05). However, a statistically significant difference in allele and genotype frequencies of the –1793G/C was proved between all NIDDM versus nondiabetic subjects (p = 0.024 and 0.0065, respectively) with allele G and genotype GG being significantly more frequent in the NIDDM group. Calculated odds ratio for the GG genotype was 1.20 (95% CI, 0.77–1.86). Although significantly higher plasma von Willebrand factor antigen levels in NIDDM patients with PDR have been described in several studies, our findings indicate that no association exists between the two polymorphisms and PDR. However, the –1793G/C polymorphism might affect the risk of NIDDM.

Three novel polymorphisms in the promoter region of the TIMP-3 gene are not associated with proliferative diabetic retinopathy in type 2 diabetes mellitus.

Purpose. Tissue inhibitor of metalloproteinase-3 (TIMP-3) is a member of the TIMP family of proteins, playing a significant role in the control of extracellular matrix remodelling. TIMP-3 might play a role in the regulation of retinal neovascularization during progression of diabetic retinopathy. Recently, three novel polymorphisms (-899T/A, -915A/G and -1296T/C) in the promoter region of the TIMP-3 gene have been identified. The aim of the study was to investigate a possible association of these polymorphisms with proliferative diabetic retinopathy (PDR) in type 2 diabetes mellitus (T2DM). Methods. Genotypes were detected by polymerase chain reaction and subsequent restriction with specific endonucleases. Allele frequencies were determined in an association study comprising three groups of subjects (n = 371). Results. Linkage disequilibrium was found among the three polymorphisms (P = 0.01). Allele frequencies did not differ between neither T2DM + PDR and T2DM non-PDR subjects (P > 0.05) nor between all T2DM versus non-diabetic subjects (P > 0.05). Conclusions. Polymorphisms in the promoter region of the TIMP-3 gene were not associated with the PDR in the Caucasian T2DM patients.