Association of a functional inducible nitric oxide synthase promoter variant with complications in type 2 diabetes

Abstract

Complications of diabetes have a genetic influence. Since increased inducible nitric oxide synthase (iNOS) gene ( NOS2A) expression can contribute to tissue damage, NOS2A is a worthy candidate for such a role. We therefore tested a 4-bp insertion/deletion (+/-) polymorphism 0.7 kb upstream of NOS2A for association with complications in type 2 diabetes patients, and also performed transient transfection experiments to examine the effect of this variant on promoter activity in kidney cells in culture. We investigated 379 Caucasian type 2 diabetes patients of British/European descent, 93 of whom had microalbuminuria, 26 overt nephropathy, 46 retinopathy, and 73 clinical neuropathy. Genotyping for the variant was carried out by PCR and automated Genescan analysis. Transient transfection studies involved the renal HEK 293 cell line and luciferase reporter gene constructs containing 1.1 kb of 5'-flanking DNA from '+' or '-' allele homozygotes. We found that the '+' allele frequency in patients without microalbuminuria was 12%, but was 23% in those with microalbuminuria ( P=0.0005), and was 26% in those with nephropathy ( P=0.0007), 22% in those with retinopathy ( P=0.037), and 23% in those with neuropathy ( P=0.045). The odds ratios for homozygote +/+ to have microalbuminuria or nephropathy were 2.4 (95% CI 1.4-4.2, P=0.0023) and 5.4 (95% CI 1.8-16, P=0.0009), respectively. Luciferase reporter gene constructs containing 1 kb of NOS2A promoter DNA for each allele were made and sequence analysis confirmed that the +/- variation was the only sequence difference present. Transient transfection of these into HEK 293 cells revealed 25 times higher reporter gene activity for the '+' allele compared with the '-' allele. Gel shift analysis with 30mer oligonucleotides corresponding to each allele showed specific binding to nuclear extracts, being greater for the '+' allele. Thus the '+' allele of the NOS2A promoter variant may confer higher iNOS expression, and could contribute to complications of type 2 diabetes, especially in the approximately 5% of patients homozygous for this variant.

Proteomic Modification by Nitric Oxide

“…The activation of NOS-2 and the subsequent production of large amounts of free radical gas NO is an important anti-infectious and anti-tumor mechanism of innate immunity. However, overproduction of NO has been implicated in several pathological conditions that include (but are not limited to) the following: 1) tissue injury and various inflammatory disorders (11,12); 2) neuronal disease (13,14); 3) auto-immune diseases (15, 16); 4) cancer or tumor cell proliferation (17, 18); 5) angiogenesis and related pathological changes (19,20); and 6) diabetes mellitus (21,22).…”

An investigation of NOS2A promoter polymorphisms in Australian multiple sclerosis patients

“…is a predominantly transcriptionally regulated gene 21 -24 and promoter variants have been associated with various diseases, including complications in type II diabetes, 25 outcome of hepatitis C virus infection, 26 cerebral malaria 27,28 and dementia. 29 Two independent studies have analysed a pentanucleotide repeat in the promoter region for association with MS. 30,31 Neither study found evidence for an association between the repeat and MS in relatively small cohorts.…”

Comparison between erythrocyte hemoglobin and spectrin glycosylation and role of oxidative stress in type-2 diabetes mellitus

“…Further, these values were significantly higher in patients with complications as compared to patients without complications. Abnormal nitric oxide metabolism is related to advanced diabetic complications in type-2 diabetes mellitus, which may be the consequence of increase in inducible nitric oxide synthase gene expression (22).…”