Inducible but not endothelial nitric oxide synthase polymorphism is associated with susceptibility to rheumatoid arthritis in northwest Spain
M. A. Gonzalez-Gay*,
J. Llorca1,
E. Sanchez2,
M. A. Lopez-Nevot3,
M. M. Amoli4,
C. Garcia-Porrua,
W. E. R. Ollier4 and
J. Martin2,*
Division of Rheumatology, Hospital Xeral-Calde, Lugo, 1 Division of Preventive Medicine and Public Health, School of Medicine, University of Cantabria, Santander, 2 Instituto de Parasitologia y Biomedicina Lopez-Neyra, CSIC, Granada, 3 Division of Immunology, Hospital Virgen de las Nieves, Granada, Spain and 4 Centre for Integrated Genomic Medical Research, School of Epidemiology and Health Sciences, University of Manchester, Manchester, UK.
Correspondence to: M. A. González-Gay, Division of Rheumatology, Hospital Xeral-Calde, c/ Dr Ochoa s/n, 27004, Lugo, Spain. E-mail: miguelaggay{at}hotmail.com
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Abstract
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Objective. To assess the influence of inducible and endothelial nitric oxide synthase (iNOS and eNOS) polymorphisms in susceptibility to rheumatoid arthritis (RA).
Methods. Two hundred RA patients fulfilling the 1987 American College of Rheumatology classification criteria followed at the out-patient rheumatology clinic of the Hospital Xeral-Calde (Lugo, Spain) and 251 ethnically matched controls were studied. Patients and controls were genotyped by PCR-based techniques for a multiallelic (CCTTT)n repeat in the promoter region of the iNOS gene and for a T/C polymorphism at position 786 in the promoter region and a polymorphism in exon 7 (298Glu/Asp or 5557G/T) of the eNOS gene.
Results. No significant difference in allele or genotype frequencies for either polymorphism in the eNOS gene was observed between RA patients and controls. The overall iNOS CCTTTn allelic or genotypic distribution did not show statistical significant differences between RA patients and controls. Interestingly, when we stratified the iNOS alleles into short (811) and long (1216) repeats, significant differences were observed between RA patients and controls (P = 0.021; odds ratio = 1.37, 95% confidence interval 1.041.81). Of note, individuals carrying two alleles with a repeat number less than 12 (fewer than 196 base pairs) exhibited a double risk of developing RA (P = 0.005, odds ratio 2.26, 95% confidence interval 1.254.08).
Conclusions. Significant differences in the iNOS promoter polymorphism genotype frequency between northwest Spanish RA patients and controls suggest a potential role for this polymorphism in susceptibility to RA.
KEY WORDS: Rheumatoid arthritis, Disease susceptibility, Rheumatoid factor, Genetics, Nitric oxide synthases, Polymorphism
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Introduction
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Several studies have suggested that genes within the major histocompatibility complex rheumatoid arthritis (RA) account for only one-third to one-half of the total genetic contribution [1, 2]. Thus, the assessment of the potential influence of other genes in the development of RA is of main importance in improving our understanding of susceptibility to this condition.
Several lines of evidence indicate that nitric oxide (NO) may be important in the pathogenesis of RA [3]. Peripheral blood mononuclear cells from RA patients have increased expression of inducible NO synthase (iNOS) and enhanced formation of NO that correlates with disease activity [4, 5]. In addition, NO has been shown to be a key mediator of apoptosis within RA joints [6] and an important regulator of the Th1/Th2 balance in autoimmune diseases [7].
NO is produced constitutively by endothelial (eNOS or NOS3) or neuronal synthases (nNOS or NOS1) and, in higher concentrations, by iNOS (or NOS2) after stimulation by a variety of proinflammatory cytokines [8]. Several functionally relevant polymorphisms in the iNOS and eNOS genes have been identified, which have been associated with different vascular [9], autoimmune [10] and infectious [11] diseases. On this basis, the human iNOS and eNOS genes may be potential candidates for genetic association with RA.
A highly polymorphic pentanucleotide (CCTTT)n repeat located in the iNOS gene promoter region has been shown to be functionally important in the regulation of iNOS transcription [10], which may explain the differences observed in iNOS expression and NO formation between RA patients and control subjects [4]. Interestingly, a recent study has shown a trend for association of variation in the (CCTTT)n repeat with RA [12].
Because of this, we sought to assess the contribution of the highly polymorphic pentanucleotide (CCTTT)n within the iNOS promoter region to susceptibility to RA in our population, and to analyse the contributions to RA of the functional eNOS gene polymorphisms at 786 and exon 7.
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Patients and methods
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Study population
The study group included a series of patients (n = 200) attending the out-patient rheumatology clinic of Hospital Xeral-Calde, Lugo, Spain. All of them met the 1987 American College of Rheumatology classification criteria for RA [13] and were treated by the same group of rheumatologists (M.A.G.-G. and C.G.-P.). Patients and ethnically matched controls (n = 251) were from the Lugo region in Galicia, northwest Spain. All individuals were of Caucasoid origin. RA patients were considered as seropositive (n = 154) if they were positive for rheumatoid factor (by nephelometry) at least twice during the course of the disease.
Each subject's written consent was obtained according to the Declaration of Helsinki, and the design of the work was approved by the Ethical Committee of Galicia (Spain).
Detection of eNOS polymorphisms
The eNOS 786 and exon 7 variations were analysed by polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) as described [14]. Briefly, for determination of the 786 polymorphism, a 282-base pair fragment was amplified using the following primers: forward 5'-GTGTACCCCACC TGCATT CT-3' and reverse 5'-CCCAGCAAGGATGTAGTGAC-3'. The amplified fragment was digested with the enzyme MspI, resulting in products of 194 and 88 base pairs for allele T and 149, 88 and 45 base pairs for allele C.
For determination of the exon 7 polymorphism, a 248-base pair fragment was amplified using the following forward and reverse primers: 5'-AAG GCA GGA GAC AGT GGA TGGA-3' and 5'-CCC AGT CAA TCC CTT TGG TGC TCA-3' respectively. The PCR product was digested with the enzyme BanII, which recognizes the G allele.
Detection of iNOS (CCTTT)n polymorphism
PCR combined with fluorescence technology was used for (CCTTT)n genotyping, as previously described [15].
Statistical analysis
Strength of association between RA and alleles or genotypes of polymorphisms in the iNOS and eNOS genes was estimated using odds ratios (OR) and 95% confidence intervals (CI). Levels of significance were determined using contingency tables by either
2 or Fisher exact analysis. Statistical significance was defined as P
0.05. Calculations were performed with the statistical package Stata V6.
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Results
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eNOS gene polymorphisms in RA
eNOS gene polymorphisms, comprising a T/C polymorphism at position 786 in the promoter region and a polymorphism in exon 7 (298Glu/Asp), were examined in patients with RA. No significant differences in allele and genotype frequencies between patients and controls were observed (Table 1). Likewise, no associations were observed when RA patients were stratified by the presence of rheumatoid factor (data not shown).
iNOS promoter CCTTT repeat microsatellite polymorphism in RA
The CCTTTn allele and genotype frequencies were examined in RA patients and controls. The overall CCTTTn allelic and genotypic distributions did not show statistically significant differences between RA patients and controls (Table 2a and b). Next, we analysed the possible contribution of the 10-repeat single allele (186 base pairs), previously reported to be increased in RA compared with a control population [12], to RA predisposition in our northwest Spanish population. The 10-repeat allele frequency showed a trend of association (P = 0.08; OR = 1.45, 95% CI 0.932.28), although it did not reach statistical significance.
Interestingly, when we stratified the iNOS alleles into short (811) and long (1216) repeats, significant differences were observed between RA patients and controls (P = 0.021; OR = 1.37, 95% CI 1.041.81) (Table 3). Of note, individuals carrying two alleles with a repeat number less than 12 (less than 196 base pairs) exhibited a double risk of developing RA (P = 0.005, OR = 2.26, 95% CI 1.254.08) (Table 3).
No significant differences in allele or genotype distribution were found when RA patients were stratified according to rheumatoid factor status (data not shown). In addition, RA patients previously genotyped for HLA-DRB1 (n = 178) were stratified based on the presence or absence of HLA-DRB1*04 or DRB1*01 shared epitope (SE) alleles. No statistically significantly association between iNOS alleles and HLA-DRB1*SE alleles was found (data not shown).
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Discussion
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The present study constitutes the first attempt to assess the implication of two eNOS gene polymorphisms, at positions 786 in the promoter region and in exon 7 of the gene, in susceptibility to RA. The eNOS gene polymorphisms have been implicated in scleroderma, Behçet's disease and giant cell arteritis [1618]. Previous studies on eNOS polymorphisms in rheumatic diseases in the Lugo population of northwest Spain have shown contradictory results. In this regard, although haplotype association with biopsy-proven giant cell arteritis was found [14], no genetic effect of the eNOS polymorphisms in susceptibility to cutaneous vasculitis that fulfilled classification criteria for HenochSchönlein purpura was observed [19]. Our present data do not support the implication of the eNOS polymorphisms in susceptibility to RA. These observations suggest that the polymorphism of genes other than the eNOS gene may be responsible for the expression of cytokines, and proinflammatory mediators such as NO, which are implicated in the inflammatory response and disease susceptibility. In support of our results, the eNOS gene has been mapped to chromosome 7, within a region that has not previously been genetically linked to RA.
Some studies have yielded evidence of association of the iNOS2 promoter polymorphism with severe clinical manifestations of insulin-dependent diabetes mellitus [10, 20]. However, we could not detect an association of the CCTTTn variation with the RA severity markers analysed, the presence of the SE, or rheumatoid factor.
Our data confirm the trend of association observed in a particular population of southern Spain between the 10-repeat allele (186 base pairs) and RA [12]. It is worth noting the significantly increased frequency of short-repeat alleles observed in our series of RA patients (43%) compared with controls (35.5%); furthermore a double risk of developing RA was observed in individuals carrying two alleles with a repeat number less than 12. Since NO is an important regulator of Th1/Th2 balance, limiting the Th1 response, a possible explanation for this association may be that individuals carrying two alleles with fewer than 12 repeats (low NO producers) [10] may be susceptible to increased predisposition to autoimmune diseases. Consistent with our data, the iNOS gene has been mapped on chromosome 17q, and a region on this chromosome 17q has been shown to contain an RA susceptibility allele [21]. Therefore, it is possible that the potential role of short-repeat alleles may be due to other polymorphisms in linkage disequilibrium with these CCTTTn alleles located on chromosome 17q.
Further replication studies in different populations are necessary to fully establish the role of this iNOS CCTTT'n polymorphism in RA and its relationship with other genes implicated in RA susceptibility.
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Acknowledgments
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We thank Sonia Morales for excellent technical assistance.
J.M. has declared that this work was supported by grant SAF03-3460 from Plan Nacional de I+D+I, and in part by Junta de Andalucía (Spain). The other authors have declared no conflicts of interest.
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Notes
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*Drs Gonzalez-Gay and Martin share senior authorship in this study. 
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References
|
---|
- Deighton CM, Walker DJ, Griffiths ID, Roberts DF. The contribution of HLA to rheumatoid arthritis. Clin Genet 1989;36:17882.[ISI][Medline]
- Ollier WE, Harrison B, Symmons D. What is the natural history of rheumatoid arthritis? Baillieres Best Pract Res Clin Rheumatol 2001;15:2748.[CrossRef][ISI]
- Clancy RM, Amin AR, Abramson SB. The role of nitric oxide in inflammation and immunity. Arthritis Rheum 1998;41:114151.[CrossRef][ISI][Medline]
- St Clair EW, Wilkinson WE, Lang T et al. Increased expression of blood mononuclear cell nitric oxide synthase type 2 in rheumatoid arthritis. J Exp Med 1996;184:11738.[Abstract]
- Yki-Jarkiven H, Bergholm R, Leirisalo-Repo M. Increased inflammatory activity parallels increased basal nitric oxide production and blunted response to nitric oxide in vivo in rheumatoid arthritis. Ann Rheum Dis 2003;62:6304.[Abstract/Free Full Text]
- vant Hof RJ, Hocking L, Wright PK, Ralston SH. Nitric oxide is a mediator of apoptosis in the rheumatoid joint. Rheumatology 2000;39:10048.[Abstract/Free Full Text]
- Kolb H, Kolb-Bachofen V. Nitric oxide in autoimmune disease: cytotoxic or regulatory mediator? Immunol Today 1998;19:55661.[CrossRef][ISI][Medline]
- Weinberg JB. Nitric oxide production and nitric oxide synthase type 2 expression by human mononuclear phagocytes: a review. Mol Med 1998;4:55791.[ISI][Medline]
- Wang XL, Wang J. Endothelial nitric oxide synthase gene sequence variations and vascular disease. Mol Genet Metab 2000;70:24151.[CrossRef][ISI][Medline]
- 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;13:182532.[Abstract/Free Full Text]
- Burgner D, Xu W, Rockett K et al. Inducible nitric oxide synthase polymorphism and fatal cerebral malaria. Lancet 1998;352:11934.[ISI][Medline]
- Pascual M, Lopez-Nevot MA, Caliz R et al. Genetic determinants of rheumatoid arthritis: the inducible nitric oxide synthase (NOS2) gene promoter polymorphism. Genes Immun 2002;3:299301.[CrossRef][ISI][Medline]
- Arnett FC, Edworthy SM, Bloch DA et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:31524.[ISI][Medline]
- Amoli MM, Garcia-Porrua C, Llorca J, Ollier WE, Gonzalez-Gay MA. Endothelial nitric oxide synthase haplotype associations in biopsy-proven giant cell arteritis. J Rheumatol 2003;30:201922.[ISI][Medline]
- 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;204:16570.[CrossRef][ISI][Medline]
- Fatini C, Gensini F, Sticchi E et al. High prevalence of polymorphisms of angiotensin-converting enzyme (I/D) and endothelial nitric oxide synthase (Glu298Asp) in patients with systemic sclerosis. Am J Med 2002;112:5404.[CrossRef][ISI][Medline]
- Salvarani C, Boiardi L, Casali B et al. Endothelial nitric oxide synthase gene polymorphisms in Behcet's disease. J Rheumatol 2002;29:53540.[ISI][Medline]
- Salvarani C, Casali B, Nicoli D et al. Endothelial nitric oxide synthase gene polymorphisms in giant cell arteritis. Arthritis Rheum 2003;48:321923.[CrossRef][ISI][Medline]
- Amoli MM, Garcia-Porrua C, Calviño MC, Ollier WE, Gonzalez-Gay MA. Lack of association between endothelial nitric oxide synthase polymorphism and HenochSchonlein purpura. J Rheumatol 2004;31:299301.[ISI][Medline]
- Johannesen J, Tarnow L, Parving 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;23:5602.[Free Full Text]
- Barton A, Eyre S, Myerscough A et al. High resolution and association mapping identifies a novel rheumatoid arthritis susceptibility locus homologous to one linked to two rat models of inflammatory arthritis. Hum Mol Genet 2001;10:19016.[Abstract/Free Full Text]
Submitted 11 January 2004;
revised version accepted 28 May 2004.