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

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 Gene-scan 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.