Endothelial nitric oxide synthase (eNOS) VNTR as a probable marker in type 2 diabetes mellitus.

Background: The gene encoding eNOS is located on chromosome 7q36, a genetic region previously linked to metabolic syndrome, cardiovascular and renal diseases. Generally, in diabetes there are numerous genes involved, each being a small contributor in type 2 diabetes mellitus (T2DM) manifestation. A 27 bp variable number of tandem repeat (27 bp VNTR a/b) in intron 4 of enos gene has gained attention and this polymorphism may affect the expresssion of eNOS. We studied the association of eNOS-27 bp VNTR with T2DM in north indian population. Material and methods: Blood samples were collected in 0.5 M EDTA from 200 T2DM patients and 210 age/sex matched healthy controls. Genomic DNA was extracted from peripheral blood mononuclear cells (PBMCs) using the salting out method. The 27-VNTR polymorphism was determined by standard PCR amplification using forward and reverse primers 5'-AGGCCCTATGGTAGTGCCTTT-3’ and 5'-TCTCTTAGTGCTGTGGTCAC-3’ respectively. The genotypes were determined by analyzing the amplified products on 2% agarose gels. Genotypic and allelic frequencies were calculated by SPSS (version 15.0). Results: Clinical and biochemical profiles of healthy controls and T2DM cases as well as gender wise comparisons showed significant association in certain parameters (p<0.001). Five different alleles (I, II, IV, V and VI) were found in the study population. The genotypic frequency was significantly associated with T2DM (P <0.001). Conclusion: A significant role of allele ‘I’ in T2DM susceptibility was an interesting observation. Therefore, The 27 bp VNTR in eNOS gene polymorphism can be used as a probable marker in determining susceptibility to T2DM in north Indian population.

Interleukin-6 (IL-6)-597 A/G (rs1800797) & -174 G/C (rs1800795) gene polymorphisms in type 2 diabetes.

Background & objectives: Diabetes is a metabolic pro-inflammatory disorder characterized by chronic hyperglycaemia and increased levels of circulating cytokines suggesting a causal role for inflammation in its aetiology. In order to decipher the role of interleukin-6 (IL-6) in type 2 diabetes mellitus (T2DM) we analyzed two promoter polymorphisms -597 A/G (rs1800797) and -174 G/C (rs1800795) in T2DM cases from north India, and in healthy controls. methods: DNA was isolated from venous blood samples of T2DM patients (n=213) and normal healthy controls (n=145). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed after biochemical analysis. The genotypic and allelic frequency distributions were analyzed. Results: The clinical/biochemical parameters of T2DM cases when compared to controls showed a significant difference. No significant association was observed with -597A/G polymorphism while, -174 G/C showed a highly significant association (P<0.001). In haplotypic analysis, combination of -597G*/-174C* showed significant association (P=0.010). Interpretation & conclusions: Our data suggest that IL-6 gene polymorphisms play a prominent role in T2DM disease susceptibility in population from north India.

Reactive metabolites and antioxidant gene polymorphisms in type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM), by definition is a heterogeneous, multifactorial, polygenic syndrome which results from insulin receptor (IR) dysfunction. It is an outcome of oxidative stress caused by interactions of reactive metabolites (RMs) with lipids, proteins and other molecules of the human body. Production of RMs mainly superoxides (•O2 −) has been found in a variety of predominating cellular enzyme systems including nicotinamide adenine dinucleotide phosphate oxidase, xanthine oxidase, cyclooxygenase, endothelial nitric oxide synthase (eNOS) and myeloperoxidase. The four main RM related molecular mechanisms are: increased polyol pathway flux; increased advanced glycation end‑product formation; activation of protein kinase C isoforms and increased hexosamine pathway flux which have been implicated in glucose‑mediated vascular damage. Superoxide dismutase, catalase, glutathione peroxidase, glutathione‑S‑transferase and NOS are antioxidant enzymes involved in scavenging RMs in normal individuals. Functional polymorphisms of these antioxidant enzymes have been reported to be involved in the pathogenesis of T2DM. The low levels of antioxidant enzymes or their non‑functionality results in excessive RMs which initiates stress related pathways thereby leading to IR and T2DM. An attempt has been made to review the role of RMs and antioxidant enzymes in oxidative stress resulting in T2DM.

Preliminary studies on CD36 gene in type 2 diabetic patients from north India.

Background & objectives: The greater tendency to diabetes in Indians may be due to genetic factors in addition to environment and diet. CD36, a class B scavenger cell surface receptor mediates internalization of oxidized low density lipoprotein (Ox-LDL) leading to the formation of macrophage foam cells. CD36 deficiency is related to phenotypic expression of the metabolic syndrome, frequently associated with atherosclerotic cardiovascular diseases resulting in raised levels of glucose thereby contributing to type 2 diabetes (T2DM). Therefore, the association of human CD36 gene mutation to T2DM needs investigation. We undertook this study to investigate CD36 gene status in north Indian subjects by screening for the deletion of exons 3, 4 and 5 and certain polymorphisms. Methods: Clinical characteristics were compared between 300 T2DM patients and 100 healthy controls. Deletion analysis was carried out for exons 3, 4 and 5 of CD36 gene in 300 T2DM patients using PCR and agarose gel electrophoresis. Genotype analysis for two polymorphisms 478C>T and delAC in exons 4 and 5 respectively was carried out using PCR-RFLP method. Results: Biochemical parameters such as fasting and post-prandial glucose levels, total cholesterol, LDL-cholesterol and blood pressure were slightly raised in the T2DM patients when compared with controls with lowered HDL-cholesterol. No exonic deletion was observed in the 300 patients and 100 controls screened. All individuals were found to be homozygous (CC and -/-) for the two polymorphisms studied. Interpretation & conclusions: Although no exonic deletion was found in T2DM patients, our study suggests that all 15 exons need to be screened for mutations which lead to CD36 deficiency. Genotyping studies of the two SNPs in the CD36 gene confirmed the absence of exons 4 and 5 deletion. This is perhaps the first report from India suggesting that CD36 is one of the several important genes that need to be explored in relation to T2DM.

Vitamin D receptor (FokI, BsmI and TaqI) gene polymorphisms and type 2 diabetes mellitus : A North Indian study.

Background : The vitamin D receptor (VDR) gene is a candidate gene for susceptibility to several diseases. Studies on association between VDR polymorphisms and risk of type 2 diabetes (T2DM) in different ethnic populations are yet inconclusive. Aims : This study was conducted to evaluate association between VDR polymorphisms and genetic susceptibility to T2DM in the north Indian population. Settings and Design : One hundred clinically diagnosed T2DM patients and 160 healthy controls from the north Indian population were recruited for genetic association study. Materials and Methods : Genomic DNA was extracted from blood and genotyped for the single nucleotide polymorphism SNPs of FokI (T/C) [rs2228570], BsmI (A/G) [rs1544410] and TaqI (C/T) [rs731236] by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis. Statistical Analysis Used : Genotype distribution and allelic frequencies were compared between patients and controls. Mean values and odds ratios (ORs) with 95% confidence interval (CI) were calculated using SPSS software (version 15.0). Results : The genotype distribution, allele and haplotype frequencies of VDR polymorphism did not differ significantly between patients and controls. Mean age and waist-hip ratio of patients were found to be associated with VDR polymorphism. Combination studies showed FFBbtt increased the risk of T2DM in north Indians. Conclusions : Our data suggest that VDR gene polymorphism in combination of genotypes is associated with the risk of T2DM and thus requires further studies as a probable genetic risk marker for T2DM.

Association of IL-4 and IL-1RN (receptor antagonist) gene variants and the risk of type 2 diabetes mellitus: A study in the north Indian population.

Background: Inflammation is a key event closely associated with the pathophysiology of type 2 diabetes mellitus (T2DM). Association of genetic polymorphisms of inflammatory cytokines with T2DM is largely unknown. Our objective was to investigate the relationship of polymorphism of IL-1RN and IL-4, two important biomarkers of inflammation, with the risk of T2DM. Setting and Design: We recruited 120 clinically diagnosed T2DM patients and 150 normal healthy controls for this study in order to evaluate the nature of polymorphisms of IL-1RN and IL-4. Materials and Methods: Genomic DNA was isolated from the blood of all subjects, and the variable number of tandem repeat (VNTR) polymorphisms of IL-1RN and IL-4 genes was identified by polymerase chain reaction. Statistical Analysis Used: Genotype distribution and allelic frequencies were compared between patients and control group. Means, as well as odds ratios (ORs) with 95% confidence intervals (CI), were calculated using SPSS software (version 11.5). Results: Our study revealed that distribution of both IL-4 and IL-1RN (VNTR) gene polymorphisms were significantly associated with T2DM subjects. We, however, failed to find any association of gene-gene (IL-4 and IL-1RN) interaction with T2DM. Conclusions: Both IL-4 and IL-1RN (VNTR) gene polymorphisms were significantly associated with T2DM subjects. This may suggest that the genetic polymorphisms of IL-4 and IL-1RN genes could serve as susceptibility indicators for T2DM in the Indian population, but the actual mechanism of these associations will require more elaborate investigations. Lack of association of gene-gene (IL-4 and IL-1RN) interaction with T2DM may indicate the independent nature of influence of both these genes on the risk of T2DM.