An association study and a linkage analysis were carried out in parallel in order to investigate the association of the inducible nitric oxide synthase (NOS2) gene promoter polymorphism with rheumatoid arthritis (RA) predisposition and/or outcome including a −954G-C mutation, a di-allelic (TAAA)n marker and the highly polymorphic (CCTTT). The −954G-C point mutation occurred non-polymorphic and the di-allelic (TAAA)n marker was not associated with RA predisposition. The (CCTTT)nlocus showed a trend for RA association in the case-control study, however, stratification data by Class II status did not yield significant effect and overall, the family study showed no significant association nor linkage for any of the markers under study using TDT and non-parametric linkage respectively. Finally, no influence was detected regarding any of the clinical parameters tested. After evaluating for the first time the influence of NOS2 promoter polymorphism in RA, it seems to have no major effect on disease susceptibility and/or outcome.
Nitric oxide (NO) is constitutively produced by endothelial or neuronal NO synthases, or in higher concentrations by inducible NO synthase (iNOS; NOS2) after stimulation of bacterial products and cytokines.1 Several lines of evidence indicate that NO may be important in the pathogenesis of rheumatoid arthritis (RA).2,3 Studies in experimental animal models of arthritis have suggested that excessive levels of NO can promote tissue injury and contribute to progression of the disease.3 Likewise, it has been shown that peripheral blood mononuclear cells (PBMC) from RA patients, have increased NOS2 expression and enhanced formation of NO that correlates with disease activity4 and similarly, NO has been shown as a key mediator of apoptosis within the RA joints5 as well as an important regulator of the Th1/Th2 balance in autoinmune diseases.6 The final piece of evidence comes from the finding that administration of NOS2 inhibitors has provided beneficial effects on animal models of RA.3
On this basis we considered the human NOS2 gene as a novel candidate for genetic association with RA susceptibility and severity. Three different polymorphisms have been identified in the human NOS2 promoter region; a single nucleotide polymorphism (SNP) (G/C) at position −9547 and two microsatellite (ms) repeats; a biallelic tetranucleotide repeat sequence (TAAA)n8 and a highly polymorphic (nine alleles) pentanucleotide (CCTTT)n repeat.9 Tandem repeat variations within the promoter region may affect NOS2 transcription explaining the differences observed in NOS2 expression and NO formation between RA patients and control subjects.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.
Results and discussion
The study included 152 Spanish RA patients and 199 healthy voluntary blood donors from the Granada area (southern Spain) and detailed information on clinical parameters and HLA typing has been published elsewhere.11 Within the three NOS2 promoter polymorphisms investigated, the analysis of the 954G/C single point variation using a PCR-RFLP method,12 showed that only the wild type ‘G’ allele occurred among a first group of 50 patient and 50 control samples, therefore no further genotyping was accomplished. This is consistent with former studies where it has always been shown non-polymorphic in Caucasian populations.7,13 Regarding the bi-allelic TAAA marker (for methods see Glenn et al14) no disease association was observed in our case-control cohort (genotype percentages in RA patients vs controls; 220 bp/220 bp: 78.3% vs 70.1%, 220 bp/240 bp: 19.8% vs 28.8 and 240 bp/240 bp: 1.9% vs 1.1%). These data are in concordance with previous reports showing no association of this (TAAA)n marker with diabetes and hypertension13,14 and confirm that the (TAAA)n marker is not very informative in detecting association due to its limited degree of polymorphism. 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 pattern15 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). When we compared the distribution of the CCCTT repeat within the control subjects with that of 152 RA patients, both 10 and 15 repeat alleles (186 and 211 bp) showed a trend for increased frequency in the patients group (P = 0.01, OR 1.69, 95% CI 1.07–2.65 and P = 0.03, OR 2.52, 95% CI 0.99–6.57 respectively) that turned out nonsignificant after applying multiple testing (Pc = NS for both alleles). Influence of Class II region on RA predisposition in the population under study has been discussed elsewhere,11 showing that DQ3-DR4 individuals carried the highest risk of developing the disease. In fact, stratification data of 186 and 211 NOS2 alleles by Class II status did not yield significant associations of any of both alleles with RA. The simultaneous family study including 47 Spanish families consecutively recruited by the European Consortium on RA families (ECRAF) with a single RA affected individual,16 showed no significant association or linkage for NOS2 ms markers by both TDT and non-parametric linkage analysis respectively. Taken together, our data suggest that NOS2 gene polymorphisms might not have major effect on RA susceptibility.
Genetic susceptibility to RA is complex and heterogeneous and likely involves several polymorphic genes, perhaps each contributing with a small increment. The (TAAA)n and (CCTTT)n ms at the NOS2 promoter region have been analysed for association with the susceptibility to IDDM, other autoimmune and complex genetic condition, in which iNOS mediated NO production has been implicated, and similar to us, no linkage of the two NOS2 promoter microsatellites was observed in a large collection of families.13 Likewise, no association was identified when TAAA-CCTTT haplotypes were constructed and analysed in our RA case-control cohort. 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 retinopathy10 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. Indeed, some studies of NOS2 inhibition in experimental autoimmune diseases reported no amelioration or even an exarcebation of the autoaggression.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). 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. Nonetheless, further detailed molecular promoter studies using promoter constructs and gel retardation assays are needed to define the overall importance of NOS2 promoter polymorphisms. It could also be the case that other polymorphisms in LD might be influencing the promoter activity.
In summary, our data suggest that the NOS2 gene promoter polymorphisms do not have a major effect on RA predisposition or outcome, however it would be of interest to explore further the finding of an effect of medium CCTTT alleles on RA protection. In addition, the reported genetic heterogeneity within and between ethnic groups in the highly polymorphic promoter (CCTTT)n repeat13 raises the possibility that a (CCTTT)n allele might be associated with the disease in a different population. Detailed functional assays, epidemiological studies in different populations and searching of existence of additional polymorphisms in both the promoter region and the codon region of the NOS2 could be useful to address the potential involvement of the NOS2 gene in the genetic predisposition to RA.
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This work was supported by grant SAF00-213 from Plan Nacional de I+D (CICYT).
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