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Inducible but not endothelial nitric oxide synthase polymorphism is associated with susceptibility to rheumatoid arthritis in northwest Spain

Inducible but not endothelial nitric oxide synthase polymorphism is associated with susceptibility to rheumatoid arthritis in northwest Spain
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  Rheumatology 2004;43:1182–1185 doi:10.1093/rheumatology/keh283Advance Access publication 29 June 2004 Inducible but not endothelial nitric oxide synthasepolymorphism is associated with susceptibility torheumatoid arthritis in northwest Spain M. A. Gonzalez-Gay*, J. Llorca 1 , E. Sanchez 2 , M. A. Lopez-Nevot 3 ,M. M. Amoli 4 , C. Garcia-Porrua, W. E. R. Ollier 4 and J. Martin 2, * Objective . To assess the influence of inducible and endothelial nitric oxide synthase (iNOS and eNOS) polymorphisms insusceptibility to rheumatoid arthritis (RA). Methods . Two hundred RA patients fulfilling the 1987 American College of Rheumatology classification criteria followed at theout-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 regionof 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 observedbetween RA patients and controls. The overall iNOS CCTTT n  allelic or genotypic distribution did not show statisticalsignificant differences between RA patients and controls. Interestingly, when we stratified the iNOS alleles into short (8–11)and long (12–16) repeats, significant differences were observed between RA patients and controls ( P  ¼ 0.021; odds ratio ¼ 1.37,95% confidence interval 1.04–1.81). Of note, individuals carrying two alleles with a repeat number less than 12 (fewer than 196base pairs) exhibited a double risk of developing RA ( P  ¼ 0.005, odds ratio 2.26, 95% confidence interval 1.25–4.08). Conclusions . Significant differences in the iNOS promoter polymorphism genotype frequency between northwest Spanish RApatients and controls suggest a potential role for this polymorphism in susceptibility to RA. K EY WORDS:  Rheumatoid arthritis, Disease susceptibility, Rheumatoid factor, Genetics, Nitric oxide synthases, Polymorphism.Several studies have suggested that genes within the majorhistocompatibility complex rheumatoid arthritis (RA) accountfor only one-third to one-half of the total genetic contribution[1, 2]. Thus, the assessment of the potential influence of other genesin the development of RA is of main importance in improvingour understanding of susceptibility to this condition.Several lines of evidence indicate that nitric oxide (NO) maybe important in the pathogenesis of RA [3]. Peripheral bloodmononuclear cells from RA patients have increased expressionof inducible NO synthase (iNOS) and enhanced formation of NOthat correlates with disease activity [4, 5]. In addition, NO hasbeen shown to be a key mediator of apoptosis within RA joints [6]and animportant regulator ofthe Th1/Th2 balance in autoimmunediseases [7].NO is produced constitutively by endothelial (eNOS or NOS3)or neuronal synthases (nNOS or NOS1) and, in higher concen-trations, by iNOS (or NOS2) after stimulation by a variety of proinflammatory cytokines [8]. Several functionally relevant poly-morphisms in the iNOS and eNOS genes have been identified,which have been associated with different vascular [9], auto-immune [10] and infectious [11] diseases. On this basis, the humaniNOS and eNOS genes may be potential candidates for geneticassociation with RA.A highly polymorphic pentanucleotide (CCTTT) n  repeatlocated in the iNOS gene promoter region has been shown tobe functionally important in the regulation of iNOS transcrip-tion [10], which may explain the differences observed in iNOSexpression and NO formation between RA patients and con-trol subjects [4]. Interestingly, a recent study has shown a trendfor association of variation in the (CCTTT) n  repeat with RA [12].Because of this, we sought to assess the contribution of thehighly polymorphic pentanucleotide (CCTTT) n  within the iNOSpromoter region to susceptibility to RA in our population, andto analyse the contributions to RA of the functional eNOS genepolymorphisms at  786 and exon 7. Patients and methods Study population The study group included a series of patients ( n ¼ 200) attendingthe out-patient rheumatology clinic of Hospital Xeral-Calde, Correspondence to: M. A. Gonza ´lez-Gay, Division of Rheumatology, Hospital Xeral-Calde, c/ Dr Ochoa s/n, 27004, Lugo, Spain.*Drs Gonzalez-Gay and Martin share senior authorship in this study. 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. Submitted 11 January 2004; revised version accepted 28 May 2004.Rheumatology Vol. 43 No. 9    British Society for Rheumatology 2004; all rights reserved 1182   b  y g u e  s  t   on J   a n u a r  y1 2  ,2  0 1  5 h  t   t   p :  /   /  r h  e  um a  t   ol   o g y . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om   Lugo, Spain. All of them met the 1987 American College of Rheumatology classification criteria for RA [13] and weretreated by the same group of rheumatologists (M.A.G.-G. andC.G.-P.). Patients and ethnically matched controls ( n ¼ 251) werefrom the Lugo region in Galicia, northwest Spain. All individ-uals were of Caucasoid srcin. RA patients were considered asseropositive ( n ¼ 154) if they were positive for rheumatoidfactor (by nephelometry) at least twice during the course of thedisease.Each subject’s written consent was obtained according to theDeclaration of Helsinki, and the design of the work was approvedby the Ethical Committee of Galicia (Spain). Detection of eNOS polymorphisms The eNOS   786 and exon 7 variations were analysed bypolymerase chain reaction–restriction fragment length poly-morphism (PCR-RFLP) as described [14]. Briefly, for deter-mination of the   786 polymorphism, a 282-base pair fragmentwas amplified using the following primers: forward 5 0 -GTGTACCCCACC TGCATT CT-3 0 and reverse 5 0 -CCCAGCAAGGATGTAGTGAC-3 0 . The amplified fragment wasdigested with the enzyme  Msp I, resulting in products of 194and 88 base pairs for allele T and 149, 88 and 45 base pairs forallele C.For determination of the exon 7 polymorphism, a 248-base pairfragment was amplified using the following forward and reverseprimers: 5 0 -AAG GCA GGA GAC AGT GGA TGGA-3 0 and5 0 -CCC AGT CAA TCC CTT TGG TGC TCA-3 0 respectively.The PCR product was digested with the enzyme  Ban II, whichrecognizes 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 genotypesof polymorphisms in the iNOS and eNOS genes was estimatedusing odds ratios (OR) and 95% confidence intervals (CI). Levelsof 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 packageStata V6. Results eNOS gene polymorphisms in RA eNOS gene polymorphisms, comprising a T/C polymorphism atposition  786 in the promoter region and a polymorphism in exon7 (298Glu/Asp), were examined in patients with RA. No significantdifferences in allele and genotype frequencies between patients andcontrols were observed (Table 1). Likewise, no associations wereobserved when RA patients were stratified by the presence of rheumatoid factor (data not shown). iNOS promoter CCTTT repeat microsatellite polymorphism in RA The CCTTT n  allele and genotype frequencies were examinedin RA patients and controls. The overall CCTTT n  allelic andgenotypic distributions did not show statistically significantdifferences between RA patients and controls (Table 2a and b).Next, we analysed the possible contribution of the 10-repeat singleallele (186 base pairs), previously reported to be increased inRA compared with a control population [12], to RA predis-position in our northwest Spanish population. The 10-repeatallele frequency showed a trend of association ( P ¼ 0.08;OR ¼ 1.45, 95% CI 0.93–2.28), although it did not reach statisticalsignificance.Interestingly, when we stratified the iNOS alleles into short(8–11) and long (12–16) repeats, significant differences were T ABLE  2a. Allele frequencies of iNOS (CCTTT) n  gene polymorphism in RA and controls from northwest SpainRepeat Size RA patients ControlsNumber (base pairs)  n  (%)  n  (%) OR 95% CI  P 8 176 7 (1.7) 5 (1.0) 2.05 0.54–8.36 0.229 181 21 (5.3) 26 (5.2) 1.18 0.60–2.29 0.5910 186 49 (12.2) 44 (8.8) 1.63 1.00–2.67 0.0411 191 95 (23.7) 103 (20.5) 1.35 0.93–1.97 0.1012 196 125 (31.3) 183 (36.5) 1 (reference) – – 13 201 65 (16.3) 75 (14.9) 1.27 0.83–1.94 0.2514 206 30 (7.5) 48 (9.6) 0.92 0.53–1.57 0.7315 211 7 (1.7) 12 (2.4) 0.85 0.28–2.43 0.7516 216 1 (0.3) 6 (1.2) 0.24 0.01–2.06 0.16T ABLE  1. Allele and genotype frequencies of eNOS gene polymorphismsin RA and controls from northwest SpainRA patients ControlseNOS (exon 7) ( n ¼ 176) ( n ¼ 97)AlleleG 231 (66%) 115 (59%)T 121 (34%) 79 (41%)GenotypeGG 76 (43%) 35 (36%)GT 79 (45%) 45 (46%)TT 21 (12%) 17 (18%)eNOS (  786) ( n ¼ 170) ( n ¼ 117)AlleleT 211 (62%) 132 (56%)C 129 (38%) 102 (44%)GenotypeTT 66 (31%) 37 (32%)TC 79 (49%) 58 (50%)CC 25 (20%) 22 (19%)No statistically significant differences were observed. eNOS and iNOS polymorphisms in RA  1183   b  y g u e  s  t   on J   a n u a r  y1 2  ,2  0 1  5 h  t   t   p :  /   /  r h  e  um a  t   ol   o g y . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om   observed between RA patients and controls ( P ¼ 0.021; OR ¼ 1.37,95% CI 1.04–1.81) (Table 3). Of note, individuals carrying twoalleles with a repeat number less than 12 (less than 196 base pairs)exhibited a double risk of developing RA ( P ¼ 0.005, OR ¼ 2.26,95% CI 1.25–4.08) (Table 3).No significant differences in allele or genotype distributionwere found when RA patients were stratified according torheumatoid factor status (data not shown). In addition, RApatients previously genotyped for HLA-DRB1 ( n ¼ 178) werestratified based on the presence or absence of HLA-DRB1*04 orDRB1*01 shared epitope (SE) alleles. No statistically significantlyassociation between iNOS alleles and HLA-DRB1*SE alleles wasfound (data not shown). Discussion The present study constitutes the first attempt to assess theimplication of two eNOS gene polymorphisms, at positions   786in the promoter region and in exon 7 of the gene, insusceptibility to RA. The eNOS gene polymorphisms havebeen implicated in scleroderma, Behc¸et’s disease and giant cellarteritis [16–18]. Previous studies on eNOS polymorphisms inrheumatic diseases in the Lugo population of northwest Spainhave shown contradictory results. In this regard, althoughhaplotype association with biopsy-proven giant cell arteritiswas found [14], no genetic effect of the eNOS polymorphismsin susceptibility to cutaneous vasculitis that fulfilled classifica-tion criteria for Henoch–Scho ¨nlein purpura was observed [19].Our present data do not support the implication of the eNOSpolymorphisms in susceptibility to RA. These observationssuggest that the polymorphism of genes other than the eNOSgene may be responsible for the expression of cytokines, andproinflammatory mediators such as NO, which are implicated inthe inflammatory response and disease susceptibility. In supportof our results, the eNOS gene has been mapped to chromosome7, within a region that has not previously been genetically linkedto RA.Some studies have yielded evidence of association of the iNOS2promoter polymorphism with severe clinical manifestations of insulin-dependent diabetes mellitus [10, 20]. However, we couldnot detect an association of the CCTTT n  variation with the RAseverity markers analysed, the presence of the SE, or rheumatoidfactor.Our data confirm the trend of association observed in aparticular population of southern Spain between the 10-repeatallele (186 base pairs) and RA [12]. It is worth noting thesignificantly increased frequency of short-repeat alleles observedin our series of RA patients (43%) compared with controls(35.5%); furthermore a double risk of developing RA wasobserved in individuals carrying two alleles with a repeat numberless than 12. Since NO is an important regulator of Th1/Th2balance, limiting the Th1 response, a possible explanation for thisassociation may be that individuals carrying two alleles with fewerthan 12 repeats (low NO producers) [10] may be susceptible toincreased predisposition to autoimmune diseases. Consistent withour data, the iNOS gene has been mapped on chromosome 17q,and a region on this chromosome 17q has been shown to containan RA susceptibility allele [21]. Therefore, it is possible thatthe potential role of short-repeat alleles may be due to otherpolymorphisms in linkage disequilibrium with these CCTTT n alleles located on chromosome 17q.Further replication studies in different populations are neces-sary to fullyestablish the role of this iNOS CCTTT 0 n  polymorphismin RA and its relationship with other genes implicated in RAsusceptibility. T ABLE  2b. Genotype frequencies of iNOS (CCTTT) n  gene polymorphismin RA and controls from northwest SpainSize RA patients Controls(base pairs) ( n ¼ 200) ( n ¼ 251)176/181 0 (0%) 1 (0.4%)176/186 2 (1%) 0 (0%)176/191 0 (0%) 1 (0.4%)176/196 2 (1%) 1 (0.4%)176/201 2 (1%) 1 (0.4%)176/206 1 (0.5%) 0 (0%)176/211 0 (0%) 1 (0.4%)181/181 0 (0%) 2 (0.8%)181/186 2 (1%) 2 (0.8%)181/191 4 (2%) 4 (1.6%)181/196 8 (4%) 10 (4%)181/201 5 (2.5%) 3 (1.2%)181/206 2 (1%) 1 (0.4%)181/216 0 (0%) 1 (0.4%)186/186 7 (3.5%) 3 (1.2%)186/191 11 (5.5%) 5 (2%)186/196 12 (6%) 17 (6.8%)186/201 6 (3%) 5 (2%)186/206 2 (1%) 4 (1.6%)186/211 0 (0%) 4 (1.6%)186/216 0 (0%) 1 (0.4%)191/191 15 (7.5%) 8 (3.2%)191/196 32 (16%) 47 (18.7%)191/201 9 (4.5%) 20 (8%)191/206 6 (3%) 6 (2.4%)191/211 3 (1.5%) 4 (1.6%)196/196 17 (8.5%) 28 (11.2%)196/201 22 (11%) 26 (10.4%)196/206 11 (5.5%) 21 (8.4%)196/211 4 (2%) 2 (0.8%)196/216 0 (0%) 3 (1.2%)201/201 7 (3.5%) 7 (2.8%)201/206 6 (3%) 4 (1.6%)201/211 0 (0%) 1 (0.4%)201/216 1 (0.5%) 1 (0.4%)206/206 1 (0.5%) 6 (2.4%)The overall CCTTT n  genotypic distribution did not show statisticallysignificant differences between RA patients and controls.T ABLE  3. Association of shorter forms of iNOS (CCTTT) n  microsatellite repeats with RARA patients n  (%)Controls n  (%) OR 95% CI  P AllelesShort (8–11 repeats) 172 (43.0) 178 (34.5) 1.37 1.04–1.81 0.021Long (12–16 repeats) 228 (57.0) 324 (64.5) 0.73 0.55–0.96 0.021GenotypesShort (8–11 repeats)  /   69 (34.5) 99 (39.4) 1 – –  þ /– 90 (45.0) 126 (50.2) 1.01 0.68–1.54 0.91 þ / þ  41 (20.5) 26 (10.4) 2.26 1.25–4.08 0.005 1184  M. A. Gonzalez-Gay  et al  .   b  y g u e  s  t   on J   a n u a r  y1 2  ,2  0 1  5 h  t   t   p :  /   /  r h  e  um a  t   ol   o g y . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om   Acknowledgements 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 þ D þ I, and in part by Junta deAndalucı ´a (Spain). The other authors have declared no conflictsof interest. References 1. Deighton CM, Walker DJ, Griffiths ID, Roberts DF. The contribu-tion of HLA to rheumatoid arthritis. Clin Genet 1989;36:178–82.2. Ollier WE, Harrison B, Symmons D. What is the natural history of rheumatoid arthritis? Baillieres Best Pract Res Clin Rheumatol2001;15:27–48.3. Clancy RM, Amin AR, Abramson SB. The role of nitric oxide ininflammation and immunity. Arthritis Rheum 1998;41:1141–51.4. St Clair EW, Wilkinson WE, Lang T  et al  . Increased expression of blood mononuclear cell nitric oxide synthase type 2 in rheumatoidarthritis. J Exp Med 1996;184:1173–8.5. Yki-Jarkiven H, Bergholm R, Leirisalo-Repo M. Increased inflam-matory activity parallels increased basal nitric oxide production andblunted response to nitric oxide  in vivo  in rheumatoid arthritis. AnnRheum Dis 2003;62:630–4.6. van’t Hof RJ, Hocking L, Wright PK, Ralston SH. Nitric oxide isa mediator of apoptosis in the rheumatoid joint. Rheumatology2000;39:1004–8.7. Kolb H, Kolb-Bachofen V. Nitric oxide in autoimmune disease:cytotoxic or regulatory mediator? Immunol Today 1998;19:556–61.8. Weinberg JB. Nitric oxide production and nitric oxide synthase type 2expression by human mononuclear phagocytes: a review. Mol Med1998;4:557–91.9. Wang XL, Wang J. Endothelial nitric oxide synthase gene sequencevariations and vascular disease. Mol Genet Metab 2000;70:241–51.10. Warpeha KM, Xu W, Liu L  et al  . Genotyping and functional analysisof a polymorphic (CCTTT) n  repeat of NOS2A in diabetic retinopathy.FASEB J 1999;13:1825–32.11. Burgner D, Xu W, Rockett K  et al  . Inducible nitric oxide synthasepolymorphism and fatal cerebral malaria. Lancet 1998;352:1193–4.12. Pascual M, Lopez-Nevot MA, Caliz R  et al  . Genetic determinants of rheumatoid arthritis: the inducible nitric oxide synthase (NOS2) genepromoter polymorphism. Genes Immun 2002;3:299–301.13. Arnett FC, Edworthy SM, Bloch DA  et al  . The AmericanRheumatism Association 1987 revised criteria for the classificationof rheumatoid arthritis. Arthritis Rheum 1988;31:315–24.14. Amoli MM, Garcia-Porrua C, Llorca J, Ollier WE, Gonzalez-GayMA. Endothelial nitric oxide synthase haplotype associations inbiopsy-proven giant cell arteritis. J Rheumatol 2003;30:2019–22.15. Xu W, Liu L, Emson PC, Harrington CR, Charles IG. Evolutionof a homopurine-homopyrimidine pentanucleotide repeat sequenceupstream of the human inducible nitric oxide synthase gene. Gene1997;204:165–70.16. Fatini C, Gensini F, Sticchi E  et al  . High prevalence of polymor-phisms of angiotensin-converting enzyme (I/D) and endothelial nitricoxide synthase (Glu298Asp) in patients with systemic sclerosis. Am JMed 2002;112:540–4.17. Salvarani C, Boiardi L, Casali B  et al  . Endothelial nitric oxidesynthase gene polymorphisms in Behcet’s disease. J Rheumatol2002;29:535–40.18. Salvarani C, Casali B, Nicoli D  et al  . Endothelial nitric oxide syn-thase gene polymorphisms in giant cell arteritis. Arthritis Rheum2003;48:3219–23.19. Amoli MM, Garcia-Porrua C, Calvin ˜o MC, Ollier WE, Gonzalez-Gay MA. Lack of association between endothelial nitric oxide syn-thase polymorphism and Henoch–Schonlein purpura. J Rheumatol2004;31:299–301.20. Johannesen J, Tarnow L, Parving HH, Nerup J, Pociot F. CCTTT-repeat polymorphism in the human NOS2-promoter confers low riskof diabetic nephropathy in type 1 diabetic patients. Diabetes Care2000;23:560–2.21. Barton A, Eyre S, Myerscough A  et al  . High resolution andassociation mapping identifies a novel rheumatoid arthritis suscep-tibility locus homologous to one linked to two rat models of inflammatory arthritis. Hum Mol Genet 2001;10:1901–6.      R     h   e   u   m   a    t   o     l   o   g   y Key messages   Unlike eNOS polymorphisms, the iNOSCCTTT n  polymorphism shows signifi-cant differences in genotype frequencybetween RA and controls, suggesting apotential role for this promoter poly-morphism in susceptibility to RA.eNOS and iNOS polymorphisms in RA  1185   b  y g u e  s  t   on J   a n u a r  y1 2  ,2  0 1  5 h  t   t   p :  /   /  r h  e  um a  t   ol   o g y . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om 
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