The inducible nitric oxide synthase promoter polymorphism does not confer susceptibility to systemic lupus erythematosus

M. Á. López-Nevot, L. Ramal, J. Jiménez-Alonso1 and J. Martín2,

Servicio de Inmunología and
1 Servicio de Medicina Interna, Hospital Virgen de las Nieves and
2 Instituto de Parasitología y Biomedicina ‘López-Neyra’, CSIC, Granada, Spain


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objective. There is increasing evidence that nitric oxide (NO) may be important in the pathogenesis of systemic lupus erythematosus (SLE). One possible explanation for the differences observed in NO production between SLE patients and controls is variation in the 5' promoter region of the NOS2 gene, which controls NOS2 transcription. We studied the possible contribution of (CCTTT)n microsatellite polymorphism in the NOS2 promoter region to susceptibility to SLE and the clinical outcome of the disease.

Methods. We analysed the distribution of the multiallelic (CCTTT)n repeat within the 5' upstream promoter region of the NOS2 gene, by a polymerase chain reaction-based method, in 117 SLE patients and 199 healthy subjects from southern Spain.

Results. No statistically significant differences between SLE patients and healthy controls were observed with regard to the frequency of (CCTTT)n microsatellite repeats of any given length. Similarly, no associations were found with any of the clinical characteristics tested.

Conclusion. We conclude that polymorphism in the NOS2 gene promoter does not play a relevant role in the pathogenesis of SLE in our population.

KEY WORDS: Systemic lupus erythematosus, Nitric oxide synthase, Microsatellite polymorphism.


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Systemic lupus erythematosus (SLE) is a non-organ-specific autoimmune disorder of unknown aetiology with a large spectrum of clinical manifestations and a variety of immunological features. The disease is characterized by autoantibody production and inflammatory manifestations such as nephritis, vasculitis, arthritis and lymphadenopathy. It is known that inflammatory conditions such as SLE are associated with increased production of nitric oxide (NO), a free radical produced from L-arginine by a group of related NO synthases (NOS), which plays an important role in several biological processes [1, 2]. There is increasing evidence that NO may be important in the pathogenesis of SLE. Studies in experimental animal models of lupus have indicated that NO is a critical mediator of this autoimmune disease, and that NO synthase inhibitors can ameliorate disease manifestations [3, 4]. Recent studies have shown that NO levels are significantly more elevated in patients with SLE than in controls, and that there is a correlation between serum measures of NO production and lupus disease activity [57].

Nitric oxide is produced constitutively by endothelial (eNOS) or neuronal NO synthases and, in higher concentrations, by inducible NO synthase (iNOS, NOS2) after stimulation by bacterial products and cytokines [8]. One possible explanation for the differences observed in NO production between SLE patients and controls is variation in the 5' promoter region of the NOS2 gene, which controls NOS2 transcription. The NOS2 gene is located at chromosome 17q11.2–12. Three different polymorphisms have been identified in the human NOS2 promoter region: a single-nucleotide polymorphism (G/C) at position –954 [9] and two microsatellite repeats, viz. a biallelic tetranucleotide repeat sequence (TAAA)n [10] and a highly polymorphic (nine alleles) pentanucleotide (CCTTT)n repeat [11], which has been shown to be functionally important in the transcriptional regulation of NOS2 expression [12]. In the present study we explored the possible contribution of the (CCTTT)n microsatellite polymorphism in the NOS2 promoter region to susceptibility SLE and its clinical outcome.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients
The present study included 117 Spanish SLE patients recruited from the Hospital Virgen de las Nieves in Granada, Spain. These were compared with 199 ethnically matched, random healthy control individuals from the same geographical area. The SLE patients and controls were Spanish Caucasians, and were matched for age and sex.. The mean age of patients at diagnosis was 32±11.6 yr (range 14–51); 106 SLE patients were female (91%) and 11 were male (9%). All patients fulfilled at least four of the American College of Rheumatology criteria for the diagnosis of SLE [13]. Written informed consent was obtained from all patients. The clinical manifestations studied were articular involvement, renal involvement, cutaneous lesions, haematopoietic alterations, neurological disease and serositis (Table 1Go). In addition, clinical activity or severity was assessed, using the SLE Disease Activity Index (SLEDAI), every 6 months [14].


View this table:
[in this window]
[in a new window]
 
TABLE 1. Frequencies of clinical manifestations in patients with SLE

 

(CCTTT)n genotyping
Polymerase chain reaction (PCR)-based genotyping for (CCTTT)n was performed as described previously [11]. Forward and reverse primers were 5'-ACCCCTGGAAGCCTACAACT-3' and 5'-GCCACTGCACCCTAGCCTGTCTCA-3' respectively. The forward primer was 5'-labelled with the fluorescent dye 6-carboxyfluorescein amino hexy (6-FAM). PCR aliquots (0.5 µl) were added to 3 µl formamide and 0.5 µl internal size standard. Samples were analysed in denaturing gels (6% acrylamide/7 M urea) and sized using GenescanTM 672 software (Applied Biosystems, Foster City, CA, USA).

Statistical methods
For association studies, P values were calculated by the {chi}2 method or Fisher's exact test as appropriate. Odds ratios (OR) with 95% confidence intervals (CI) were calculated according to Woolf's method. P values were corrected for the number of alleles determined. SPSS 9.0 software (SPSS Inc., IL, USA) was used to analyse the data. For non-parametric data analysis, the Mann–Whitney U-test was used for ordinal variables and Fisher's exact test for dichotomous variables.


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
We analysed the distribution of the multiallelic (CCTTT)n repeat within the 5' upstream promoter region of the NOS2 gene in 117 SLE patients and 199 healthy subjects from southern Spain. The 11 different alleles found had 7–17 repeats and 171–121 base pairs (bp); the numbers of repeats occurring most commonly in the control population were 11 (20%), 12 (31%) and 13 (19%) (Table 2Go). The allele frequencies within the control group showed a unimodal distribution with the peak at the 12-repeat allele, identical to that found in Caucasian populations [15] but different from the bimodal curve reported for Gambian [16] and Tanzanian children and black American individuals [17]. No statistically significant differences between SLE patients and healthy controls were observed with regard to the frequencies of (CCTTT)n microsatellite repeats of any given length (Table 2Go).


View this table:
[in this window]
[in a new window]
 
TABLE 2. Frequencies of (CCTTT)n alleles in SLE patients and control subjects

 
In order to investigate the possible association of microsatellite polymorphism with disease severity, we analysed the clinical characteristics of SLE patients according to their NOS2 microsatellite alleles, and no associations were found with any of the parameters tested (data not shown).


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
This is the first study concerning SLE and genetic factors at the NOS2 locus. Within the three NOS2 promoter polymorphisms described, the 954G/C variation has always been shown to be non-polymorphic in Caucasian populations [9, 18], all individuals carrying the wild-type G allele. The diallelic (TAAA)n has been reported not to be very informative for association studies due to its low degree of polymorphism and heterozygosity [19]. Therefore, we selected the multiallelic (CCTTT)n repeat because it is a highly polymorphic marker in addition to its suggested potential to affect NOS2 transcription [12, 15], which could explain the differences observed in NOS2 expression and NO formation between SLE patients and control subjects.

Highly significant differences have been reported in NOS2 allele frequencies between ethnically diverse populations [15]. The genotyping of our cohort of 199 healthy subjects from southern Spain showed that the microsatellite alleles in the population were of the Caucasian pattern [15]. Polymorphism in the CCTTT promoter microsatellite has been analysed for linkage to other autoimmune conditions in which NOS2-mediated NO production has been implicated. In this respect, certain CCTTT alleles have been suggested to confer protection against diabetes-related pathologies such as retinopathy [12] and nephropathy in insulin-dependent diabetes mellitus patients [18], although it has been reported recently not to be associated with predisposition to rheumatoid arthritis or type 1 diabetes in family studies [19, 20]. In the present investigation, no evidence was obtained to suggest that the CCCTT repeats in the NOS2 locus are genetic factors for predisposition to SLE or its clinical outcome. Although increased levels of NO have been reported to correlate with SLE activity [57], these findings could not be confirmed in a prospective study [21], suggesting that the role of NO in the pathogenesis of SLE, as well as other human autoimmune diseases, is not fully established.

Different CCCTT repeat alleles appear to have diverse effects on the ability of the 5' upstream promoter region to act as an effective transcription regulatory element after induction with interleukin-1ß [12]. Nonetheless, caution should be exercised in extrapolating the results of in vitro experiments to the individual patient, because other factors in the disease environment may affect NO production and NO biological activity. In fact, in a recent in vivo study neither NO production nor NOS2 expression in peripheral blood mononuclear cells was found to be correlated with NOS2 promoter microsatellite polymorphism in healthy individuals [22]. Further studies are required to confirm the effect attributed to NOS2 microsatellites, but it could also be the case that other polymorphisms in linkage disequilibrium might be influencing the promoter activity. Finally, it remains possible that some of the responses attributed to NO could have been mediated by non-specific inhibition of other metabolic pathways by NO inhibitors. Therefore, it is clear that NO plays an important role in autoimmunity and inflammation [2], but the pathological 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 or to determine which isoform is responsible.

In summary, a case–control cohort study was used to evaluate for the first time the influence of NOS2 microsatellite polymorphism on the predisposition to and/or the outcome of SLE, but provided no evidence for association with the disease in the population under study. The fact of genetic heterogeneity in this highly polymorphic promoter microsatellite within and between ethnic groups raises the possibility that, in different populations, a NOS2 genotype might be associated with the disease.


    Acknowledgments
 
This work was supported by grant SAF00-213 from Plan Nacional de I+D (CICYT).


    Notes
 
Correspondence to: J. Martín, Instituto de Parasitología y Biomedicina ‘López Neyra’, CSIC, C/Ventanilla no. 11, 18001 Granada, Spain. E-mail: martin{at}ipb.csic.es Back


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

  1. Bredt DS, Snyder SH. Nitric oxide: a physiologic messenger molecule. Annu Rev Biochem 1994;63:175–95.[CrossRef][ISI][Medline]
  2. Clancy RM, Amin AR, Abramson SB. The role of nitric oxide in inflammation and immunity. Arthritis Rheum 1998;41:1141–51.[CrossRef][ISI][Medline]
  3. Weinberg JB, Granger DL, Pisetsky DS et al. The role of nitric oxide in the pathogenesis of spontaneous murine autoimmune disease: increased nitric oxide production and nitric oxide synthase expression in MRL-lpr/lpr mice, and reduction of spontaneous glomerulonephritis and arthritis by orally administered NG-monomethyl-L-arginine. J Exp Med 1994;179:651–60.[Abstract]
  4. Huang FP, Feng GJ, Lindop G, Scott DI, Liew FGY. The role of interleukin 12 and nitric oxide in the development of spontaneous autoimmune disease in MRL/MP-lpr/lpr mice. J Exp Med 1996;183:1447–59.[Abstract]
  5. Gilkeson G, Cannon C, Oates J, Reilly C, Goldman D, Petri M. Correlation of serum measures of nitric oxide production with lupus disease activity. J Rheumatol 1999;26:318–24.[ISI][Medline]
  6. Wanchu A, Khullar M, Deodhar SD, Bambery P, Sud A. Nitric oxide synthesis is increased in patients with systemic lupus erythematosus. Rheumatol Int 1998;18:41–3.[CrossRef][ISI][Medline]
  7. Belmont HM, Levartovsky D, Goel A et al. Increased nitric oxide production accompanied by the up-regulation of inducible nitric oxide synthase in vascular endothelium from patients with systemic lupus erythematosus. Arthritis Rheum 1997;40:1810–6.[Medline]
  8. Nathan C. Inducible nitric oxide synthase: what difference does it make? J Clin Invest 1997;100:2417–23.[Free Full Text]
  9. Kun JF, Mordmuller B, Lell B, Lehman LG, Luckner D, Kremsner PG. Polymorphism in promoter region of inducible nitric oxide synthase gene and protection against malaria. Lancet 1998;351:265–6.[ISI][Medline]
  10. Bellami R, Hill A. A bi-allelic tetranucleotide repeat in the promoter of the human inducible nitric-oxide synthase gene. Clin Genet 1997;52:192–3.[ISI][Medline]
  11. Xu W, Liu L, Emson PC, Harrington CR, Charles IG. Evolution of a homopurine–homopyrimidine pentanucleotide repeat sequence upstream of the human inducible nitric oxide synthase gene. Gene 1997;204:165–70.[CrossRef][ISI][Medline]
  12. Warpeha KM, Xu W, Liu L et al. Genotyping and functional analysis of a polymorphic (CCTTT)n repeat of NOS2A in diabetic retinopathy. FASEB J 1999;13:1825–32.[Abstract/Free Full Text]
  13. Tan EM, Cohen AS, Fries JF et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1982;25:1271–7.[ISI][Medline]
  14. Bombardier C, Gladman DD, Urowitz MB, Caron D, Chang CH, Committee on Prognosis Studies in SLE. Derivation of the SLEDAI. A disease activity index for lupus patients. Arthritis Rheum 1992;35:630–40.[ISI][Medline]
  15. Xu W, Humphries S, Tomita M et al. Survey of the allelic frequency of a NOS2A promoter microsatellite in human populations: assessment of the NOS2A gene and predisposition to infectious disease. Nitric Oxide 2000;4:379–83.[CrossRef][ISI][Medline]
  16. Burgner D, Xu W, Rockett M et al. Inducible nitric oxide synthase polymorphism and fatal cerebral malaria. Lancet 1998;352:1193–4.[ISI][Medline]
  17. Levesque MC, Hobbs MR, Anstey NM et al. Nitric oxide synthase type 2 promoter polymorphisms, nitric oxide production, and disease severity in Tanzanian children with malaria. J Infect Dis 1999;180:1994–2002.[CrossRef][ISI][Medline]
  18. Johannesen J, Tarnow L, Parvinng HH, Nerup J, Pociot F. CCTTT-repeat polymorphism in the human NOS2-promoter confers low risk of diabetic nephropathy in type 1 diabetic patients. Diabetes Care 2000;23:560–2.[Free Full Text]
  19. Pascual M, López-Nevot MA, Cáliz R et al. Genetic determinants of rheumatoid arthritis: the inducible nitric oxide synthase (NOS2) gene promoter polymorphism. Genes Immun 2002;3:299–301.[CrossRef][ISI][Medline]
  20. Johannensen J, Pociot F, Kristiansen OP, Karlsen AE, Nerup J, DIEGG, DSGD. No evidence for linkage in the promoter region of the inducible nitric oxide synthase gene (NOS2) in a Danish type 1 diabetes population. Genes Immun 2000;1:362–6.[CrossRef][ISI][Medline]
  21. González-Crespo MR, Navarro JA, Arenas J, Martín-Mola E, de la Cruz J, Gómez-Reino JJ. Prospective study of serum and urinary nitrate levels in patients with systemic lupus erythematosus. Br J Rheumatol 1998;37:972–7.[CrossRef][ISI][Medline]
  22. Kun JF, Mordmüller B, Perkins DJ et al. Nitric oxide 2Lambaréné (G-954C), increased nitric oxide production and protection against malaria. J Infect Dis 2001;184:330–6.[CrossRef][ISI][Medline]
Submitted 1 April 2002; Accepted 10 June 2002





This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (3)
Disclaimer
Request Permissions
Google Scholar
Articles by López-Nevot, M. A.
Articles by Martín, J.
PubMed
PubMed Citation
Articles by López-Nevot, M. A.
Articles by Martín, J.
Related Collections
Systemic Lupus Erythematosus and Autoimmunity
Immunogenetics