Pro198Leu Polymorphism in the Glutathione Peroxidase 1 Gene Contributes to Diabetic Peripheral Neuropathy in Type 2 Diabetes Patients.

Glutathione peroxidase 1 (Gpx1) is an endogenous antioxidant enzyme. The T allele of the Pro198Leu polymorphism in the Gpx1 (rs1050450, 198C > T) gene is associated with reduced enzyme activity. The aim of this study was to evaluate the association between Pro198Leu polymorphism and risk of diabetic peripheral neuropathy (DPN). We examined 1244 T2DM patients and 730 healthy controls. In the patient group, 33 % had diabetic peripheral neuropathy. All subjects were genotyped for the Gpx1 Pro198Leu polymorphism by polymerase chain reaction and restriction analysis. A significant increase in the T allele and TT genotype frequencies was observed in DPN patients compared to those without DPN (OR 1.55, 95 % CI 1.30-1.85 and 1.89, 95 % CI 1.30-2.74, respectively). The association remained significant after correction for age, disease duration, HbA1c and BMI. When distribution of T allele was compared between DPN+ and DPN- subgroups and controls, OR was 1.54 for DPN+ and 1.00 for DPN- patients. In conclusion, our findings suggest that Gpx1 Pro198Leu genotypes are significantly associated with the risk of diabetic peripheral neuropathy in patients with T2DM. The study provides new clinically relevant information regarding genetic determinants of susceptibility to diabetic neuropathy.

The effect of Toll-like receptor 4 gene polymorphism on vascular complications in type 2 diabetes patients.

OBJECTIVE: The aim of our study was to assess the association between the TLR4 Asp299Gly polymorphism and vascular complications in patients with type 2 diabetes. METHODS: We examined 1090 patients with T2DM and 716 healthy controls. All subjects were genotyped for the Asp299Gly polymorphism by polymerase chain reaction (PCR) and restriction analysis. RESULTS: The genotype frequencies of the Asp299Gly polymorphism were similar in T2DM patients and controls (p=0.512 and 0.311, respectively). The polymorphism was analyzed in subgroups of patients with macro- and microvascular complications. The distribution of genotypes was significantly different between patients with CVD and those without CVD. A significant increase of G allele frequency was observed in CVD+ patients, with odds ratio 2.06 (1.27-3.34), p=0.0035. The same effect was found when patients with diabetic retinopathy were compared with those without it (OR for G allele 2.12, 95% CI 1.43-3.12, p=0.0002). There were no statistically significant differences in genotype distribution between patients with diabetic nephropathy or neuropathy and those without these complications. CONCLUSIONS: The results of our study demonstrated that the G allele of the Asp299Gly polymorphism of the TLR4 gene is associated with increased risk of cardiovascular disease and diabetic retinopathy in type 2 diabetes patients.

Matrix metalloproteinase-2 (MMP-2) gene polymorphism and cardiovascular comorbidity in type 2 diabetes patients.

OBJECTIVE: Matrix metalloproteinases (MMPs) play an important role in pathogenesis of atherosclerosis and vascular disease. We hypothesized that MMP-2 might be a susceptibility gene for cardiovascular disease (CVD) in diabetes. The aim of this study was to evaluate the association between C(-1306)T functional polymorphism in the MMP-2 gene and risk of CVD in type 2 diabetes patients. METHODS: We examined 1090 patients with T2DM and 612 controls. All subjects were genotyped for the C(-1306)T polymorphism by polymerase chain reaction (PCR) and restriction analysis. RESULTS: A significant decrease of T allele frequency was observed in patients with CVD versus those with no CVD (OR 0.44, 95% CI 0.36-0.52, p<0.0001). In contrast, OR for CC genotype was 2.19 (1.79-2.68, p<0.0001), conferring 2-fold greater odds for CVD. When the distribution of C(-1306)T was compared in subgroups with different clinical phenotypes of CVD, patients with stroke had the lowest frequency of T allele (6% vs. 11%), compared to entire CVD+ group (p<0.05). CONCLUSIONS: T2DM patients carrying the T allele of MMP-2 C(-1306)T polymorphism have a significantly reduced risk of CVD. The C(-1306)T polymorphism is associated with susceptibility to stroke in T2DM patients.

Common polymorphism in the cannabinoid type 1 receptor gene (CNR1) is associated with microvascular complications in type 2 diabetes.

Endocannabinoids exert their biological effects via interaction with G-protein coupled cannabinoid receptors CB1 and CB2. Polymorphisms in the CNR1 gene (encoding CB1 receptor) were previously found to be associated with dyslipidemia and cardiovascular diseases. We investigated a role of the polymorphism in CNR1 gene in type 2 diabetes and its complications. The study involved 667 T2DM patients and 450 healthy individuals. All subjects were genotyped for G1359A polymorphism by PCR-RFLP procedure. Genotype frequencies did not differ significantly between patients and controls. The statistically significant differences were seen between T2DM patients with diabetic nephropathy (DN) and those without it (OR for risk allele 2.84, 95% CI 2.04-3.94, p<0.0001). There were also differences between patients with diabetic retinopathy (DR) and those without DR (OR for risk allele 1.81, 95% CI 1.30-2.53, p=0.0005). No differences were observed in diabetic neuropathy. The A allele was more frequent in patients with coexisting cardiovascular disease (CVD) compared to patients without CVD (p=0.0044). The novel finding of our study is the association of the G1359A polymorphism with diabetic nephropathy and diabetic retinopathy in patients with T2DM. This polymorphism was also associated with cardiovascular disease in the patient group.

Chemotactic cytokine receptor 5 gene polymorphism: relevance to microvascular complications in type 2 diabetes.

We investigated the involvement of chemotactic cytokine receptor 5 (CCR5) gene polymorphism in microvascular complications of T2DM. All subjects were genotyped with the 59029 SNP in the CCR5 gene. The genotype/allele frequencies did not differ between T2DM patients and controls. Genotype distribution was compared in patients with and without complications (nephropathy, retinopathy and neuropathy). The frequency of A allele was significantly higher in patients with complications (OR for A allele 3.07, 95% CI 2.49-3.77). The A allele carriage was associated with diabetic nephropathy (OR 6.17, 95% CI 3.28-11.6). An association was observed between 59029 polymorphism and age at T2DM onset. The A allele was more frequent in early onset than in late onset patients. For AA homozygotes OR was 2.38 (1.19-4.76) and 2.26 (1.12-4.58) in complicated and uncomplicated subgroups, respectively. These results suggest that CCR5 gene polymorphism is associated with diabetic nephropathy in T2DM.

Complement receptor 1 gene polymorphism and cardiovascular disease in dialyzed end-stage renal disease patients.

Inflammation plays an important role in cardiovascular disease (CVD). The complement system is a critical component of innate and acquired immunity. We investigated whether the polymorphisms in the complement receptor 1 (CR1) gene are associated with CVD in end-stage renal disease (ESRD) patients. The study groups of 1200 patients with ESRD, 360 patients with type 2 diabetes and 924 healthy individuals were genotyped. The GG genotype of the C5507G polymorphism was significantly more frequent in ESRD patients with CVD than in patients without CVD and controls (odds ratio [OR] = 3.44, 95% confidence interval [CI] = 2.23-5.3, and OR = 5.46, 95% CI = 3.72-8.0, respectively). The GG genotype was observed in 62% of patients with a history of myocardial infarction. The frequency of the G allele was also higher in patients with CVD (OR = 2.24, 95% CI = 1.93-2.61 vs controls, and OR = 1.97, 95% CI = 1.63-2.36 vs patients without CVD). In the multivariate logistic regression analysis the carrier status of G allele of C5507G polymorphism was an independent risk factor of CVD in ESRD patients (p < 0.001). In conclusion, our results suggest strong association between the CR1 gene polymorphism and CVD in ESRD patients.

Heat-shock protein gene polymorphisms and the risk of nephropathy in patients with Type 2 diabetes.

HSPs (heat-shock proteins) are molecular chaperones synthesized under stress conditions, and are involved in renal cell survival and matrix remodelling in acute and chronic renal diseases. In the present study, we investigated whether the HSP70 gene polymorphisms affect susceptibility to DN (diabetic nephropathy) in patients with T2DM (Type 2 diabetes mellitus). The study group consisted of 452 patients with nephropathy. Two control subgroups involved 340 healthy individuals and 132 patients with T2DM lasting > or =10 years who were free of nephropathy. Subjects were genotyped for the HSP70-1 +190 G/C and -110 A/C, HSP70-2 +1267 A/G and HSP70-hom +2437 T/C polymorphisms by PCR, followed by digestion with restriction endonucleases. There were no statistically significant differences in genotype distribution between patients with T2DM with DN and controls for the HSP70-hom polymorphism. Significant differences were observed for HSP70-1 and HSP70-2 polymorphisms. CC homozygotes of the -110 and +190 HSP70-1 polymorphisms were more frequent in patients with T2DM with DN compared with healthy controls (22 compared with 6% and 15 compared with 6.5% respectively; P<0.01). The OR (odds ratio) for the risk allele was 2.17 [95% CI (confidence interval), 1.73-2.72] for the -110 A/C and 1.74 (95% CI, 1.40-2.15) for +190 G/C polymorphisms. A strong association with DN was found for the +1267 HSP70-2 polymorphism. The GG genotype and the G allele were associated with DN, with the OR for the G allele being 4.77 (95% CI, 3.81-5.96). All GG homozygotes in the patient group had higher LDL (low-density lipoprotein)-cholesterol levels than AA homozygotes (P<0.01), suggesting that the observed effect might be associated with this cardiovascular risk factor. These patients progressed faster to end-stage renal failure than those with other genotypes. In conclusion, our results indicate that the HSP70-1 and HSP70-2 polymorphisms are associated with renal complications in T2DM and may be useful in identifying patients with increased risk of DN.

Tumor necrosis factor-alpha gene polymorphism correlates with cardiovascular disease in patients with end-stage renal disease.

BACKGROUND AND OBJECTIVE: Tumor necrosis factor-alpha (TNFalpha) is a potent proinflammatory cytokine. Through its effects on lipid metabolism and endothelial function, TNFalpha is involved in cardiovascular disease (CVD). We have studied two polymorphisms in the promoter region of the TNFalpha gene (TNF -308G/A and TNF -238G/A) in end-stage renal disease (ESRD) patients with and without CVD. The aim was to assess the association of these polymorphisms with ESRD and cardiovascular comorbidity in hemodialyzed patients. METHODS: A total of 603 patients with ESRD treated with hemodialysis (382 patients with CVD) and 325 healthy control subjects were genotyped for the TNF -308G/A and TNF -238G/A ploymorphisms by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) procedure. RESULTS: The A allele of the TNF -308 polymorphism was more frequent in the ESRD group than in control individuals. The odds ratio (OR) for the risk allele was 2.05 (95% CI 1.48, 2.84). In the subgroup of ESRD patients with CVD, the OR was 5.76 (95% CI 3.67, 9.03) relative to ESRD patients without CVD. There was no association observed between the TNF -238 polymorphism and renal failure or CVD in ESRD patients. CONCLUSION: Our results demonstrate for the first time that the A allele of the TNF -308 polymorphism is associated with CVD in hemodialyzed ESRD patients. If confirmed in prospective studies, it may be a predictor of increased susceptibility to CVD in these patients.

Apoptosis of lymphocytes and sialic acid plasma level in patients undergoing cardiac surgery.

Increasing evidence shows that extensive tissue trauma and surgical stress are related to physical alterations of cells and cell death. It was previously reported that total sialic acid (SA) plasma concentration is elevated in patients undergoing coronary artery surgery. Shedding or secreting of SA from the cell membrane surface or releasing intracellular SA may induce apoptosis. It is possible that the terminal SA residues of carbohydrate moieties facilitate recognition and removal of apoptotic cells by phagocytes. The aim of the present study was to estimate the dynamic changes in rate ofapoptosis oflymphocytes and total sialic acid plasma level during coronary artery surgery. In 17 patients undergoing coronary artery bypass grafting surgery plasma total SA concentration was measured and the percentage of apoptotic lymphocytes was determined before operation, after aorta clumping, after the end of operation and at 6, 18, 30 and 48 h after operation. Plasma total SA concentration decreases after aortic clumping and then increases gradually during a 48 hr observation period. The percentage of apoptotic cells increases during and after surgery with the exception of a sample taken at 18 hours after operation. The findings indicate the bimodal character of apoptosis and dynamic increase in total SA plasma level, which may be considered a result of mechanical damage taken place during operation or inflammatory response to surgical trauma.

Activation of chymotrypsin-like serine protease(s) during apoptosis detected by affinity-labeling of the enzymatic center with fluoresceinated inhibitor.

There is evidence in the literature that serine (Ser) proteases, like caspases, are activated during apoptosis. Little is known, however, about individual Ser proteases and the mechanism of their activation. In the present study, we employed a new type of cell permeant reagent to detect activation of chymotrypsin-like proteases in human leukemic HL-60 cells induced to undergo apoptosis. The reagent, 5(6)-carboxyfluoresceinyl-L-phenylalanyl-chloromethyl ketone (FFCK), is a fluorochrome-labeled analog of N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), the inhibitor known to specifically and covalently bind to the active center of chymotrypsin-like enzymes. In cultures treated with the DNA topoisomerase I inhibitor, camptothecin (CPT), or tumor necrosis factor (TNFalpha), populations of cells appeared that had the capability to bind FFCK. Most FFCK-binding cells were identified by fluorescence microscopy and laser scanning cytometry (LSC) as the cells undergoing apoptosis. Frequency of cells binding FFCK strongly correlated with frequency of cells having activated caspases (r=0.98 in CPT-treated, and r=0.99 in TNFalpha-treated cultures). The observed induction of FFCK binding we interpret as representing the activation of a chymotrypsin-like apoptotic Ser protease(s). Pretreatment of cells with the poly-caspase inhibitor, Z-VAD-FMK, prevented the activation of these Ser protease(s). Pretreatment with TPCK, however, had a less pronounced, although distinct and reproducible suppressive effect, on caspase activation. The data, thus, suggest that activation of caspases is an upstream event required for activation of Ser protease(s). Activation of the latter, however, appears to additionally amplify, in a cascade-like mode, caspases activation. Differential color fluorochrome-labeling allowed us to discriminate, within the same cells, between the activation of active caspases and Ser protease(s). Despite a certain degree of co-localization, the inter- and intra-cellular pattern of caspase- vs. Ser-protease(s) was different. Our approach makes it possible to simultaneously monitor activation of caspases and Ser proteases in the same live cells that are induced to apoptosis.