Genotyping and functional analysis of a polymorphic (CCTTT)_nrepeat of NOS2A in diabetic retinopathy

Abstract

Accumulating evidence shows that the severity and rapidity of onset of diabetic retinopathy are influenced by genetic factors. Expression of the nitric oxide synthases is altered in the retinal vasculature in the early stages of diabetic retinopathy. We analyzed the allele distribution of a polymorphic pentanucleotide repeat within the 5' upstream promoter region of the NOS2A gene in samples of diabetic patients. In diabetic patients from Northern Ireland, the 14-repeat allele of the NOS2A marker was significantly associated with the absence of diabetic retinopathy. Carriers of this repeat had 0.21-fold the relative risk of developing diabetic retinopathy than noncarriers of this allele. They also had significantly fewer renal and cardiovascular complications. The ability of differing numbers of (CCTTT)(n) pentanucleotide repeats to induce transcription of the NOS2A gene was analyzed using a luciferase reporter gene assay in transfected colonic carcinoma cells. Interleukin 1beta (IL-1beta) induction was most effective in constructs carrying the 14-repeat allele. When cells were incubated in 25 mM glucose to mimic the diabetic state, IL-1beta induction was inhibited in all cases, but to a significantly lesser extent with the 14-repeat allele. These unique properties of the 14-repeat allele may confer selective advantages in diabetic individuals, which may delay or prevent microvascular complications of diabetes.

Genetic determinants of rheumatoid arthritis: the inducible nitric oxide synthase (NOS2) gene promoter polymorphism

“…Of note no linkage disequilibrium (LD) was found between the two markers. Furthermore, we did not observe any relationship between the NOS2 variants and the clinical characteristics of RA, however, there are some data showing associations of the NOS2 promoter polymorphism with severe clinical manifestations of IDDM, such as retinopathy 10 and nephropathy. 17 Several reports have shown that NO overproduction plays an important role in autoimmunity and inflammation, 3 but the pathologic processes involved are complex and studies reported so far have been unable to assess the relative importance of NO in relation to other cytokine mediators.…”

“…The multiallelic (CCTTT) n repeat is on the contrary an attractive disease-causing candidate, being a highly polymorphic marker with a recently suggested effect in NOS2 transcription. 10 Highly significant differences have been reported in the CCTTT allele frequencies between ethnically diverse populations. 15 The observed frequencies of this marker in our cohort of 199 healthy subjects from southern Spain fell within the Caucasian pattern 15 showing 11 different alleles (repeat and bp ranges 7-17 and 171-221 respectively) in unimodal distribution with the peak at the 12 repeat allele (Figure 1).…”

“…6 Accordingly, it has been recently suggested that NO is not just a cytotoxic molecule but also an important regulator of the Th1/Th2 balance limiting the Th1 response, 6 which is consistent with the observation that mice with a disrupted NOS2 gene exhibit enhanced Th1 reactivity. 18 Although single alleles have been formerly associated to disease predisposition in several disorders, 10,19 we analysed the possible effect of category alleles defined by length on RA predisposition, specially given the fact that functional studies have reported that medium repeat alleles might be associated to high NO production. 10 It is worth noting that when we divided the NOS2 alleles in low, medium and high repeat number, medium repeats alleles were significantly related to RA protection (data not shown).…”

“…18 Although single alleles have been formerly associated to disease predisposition in several disorders, 10,19 we analysed the possible effect of category alleles defined by length on RA predisposition, specially given the fact that functional studies have reported that medium repeat alleles might be associated to high NO production. 10 It is worth noting that when we divided the NOS2 alleles in low, medium and high repeat number, medium repeats alleles were significantly related to RA protection (data not shown). The proposed role of NO limiting Th1 responses would be consistent with the speculation that carriers of the high NO producing medium alleles might be protected from a damaging Th1 bias.…”

“…4 With regard to this, variation in the number of CCTTT repeats has been shown to be functionally important in the regulation of NOS2 transcription. 10 The purpose of the present study was to address the possible contribution of the described polymorphism located within the NOS2 promoter region to the susceptibility and/or severity of RA.…”