Clinical and Experimental Rheumatology 2002; 20: 829-832. BRIEF PAPER TNF+489 polymorphism ABSTRACT telomeric of the DR and DQ loci. This Objectives. To determine if a tumour region is dense with approximate- does not contribute to necrosis factor (TNF +489) polymor - ly 1 gene per 15 kilobases (kb) of DNA susceptibility to rheuma- phism is associated with susceptibility and encodes many immu n o l ogi c a l ly toid arthritis to (RA). i m p o rtant proteins including seve ra l Methods. Two European populations complement proteins, the 70 kd heat A.S. Low1, M.A. Gonzalez- were studied: 217 controls and 238 pa - shock protein, and at the telomeric end, Gay2, M. Akil1, R.S. Amos1, tients from the north of England and ap p rox i m at e ly 900 kb telomeric of 145 controls and 179 patients fro m D R B 1 , the tumour necrosis fa c t o r 1 1 D.E. Bax , C. Cannings , Spain. HLA-DRB1 and T N F + 4 8 9 (TNF) cluster containing the for A. Hajeer3, S.H. Till1, markers were typed using polymerase TNF, lymphotoxin a and b (5). In view J. Winfield1, W.E. Ollier3, chain reaction based methods. of the gene location and biologi c a l A.G. Wilson1 R e s u l t s . S t rong associations we re activities of TNF there has been intense demonstrated with shared epitope (SE) e ffo rt to determine if poly m o rp h i s m encoding HLA-DRB1 alleles in the within the TNF gene contributes to the 1Division of Genomic Medicine, Royal Hal- lamshire Hospital, Sheffield, UK; 2Division English (OR= 2.9 [2.2-3.9]) and Span - genetic background of RA. of Rheumatology, Hospital Xeral-Calde ish (OR= 2.3 [1.6-3.3]) populat i o n s , A number of single nucleotide poly- Lugo, Lugo, Spain; 3ARC Epidemiology however no association was found with m o rphisms (SNPs) have been identi- Unit, School of Epidemiology & Health TNF+489 alleles. Furt h e rm o re car - fied within the TNF promoter region at Sciences, Manchester University Medical riage of TNF+489A was not associated -1031, -862, -856, -574, -376, -308 and School, Manchester, UK. with the presence of radiological ero - -238. Some of these have been associ- Audrey S. Low, BMedSci; Miguel A. Gon- sions, rheumatoid nodules or rheuma - ated with altered gene function by some, zalez-Gay, MD; Mohammed Akil, MD; toid factor. but not all groups (6,7). The TNF -308 Rodney S. Amos, MB; Deborah E. Bax, Conclusion. The role of the TNF locus variant does not appear to be associated MD; Chris Cannings, PhD; Ali Hajeer, PhD; Simon H. Till, MB; John Winfield, in the genetic back ground of RA is with susceptibility to rheumatoid arth- MB; William E. Ollier, PhD; and Anthony u n cl e a r, h oweve r, our data does not ritis (8), however, carriage of the TNF- G. Wilson, MB, PhD. support the previous reported associa - 238A allele has been associated with Audrey Low was a recipient of a BMedSci tion of the TNF+489A allele with RA p rotection from seve re fo rms of RA studentship from the Arthritis Research susceptibility or severity. (9). Two SNPs have also been identi- Campaign. fied within intron 1 of TNF:+489 (G to Please address correspondence and Introduction A Transition) and +691 (G deletion) reprint requests to: Anthony G. Wilson, S u s c eptibility to rheumatoid art h ri t i s and disease association have recently MB, PhD, Division of Genomic Medicine, (RA) is multifactorial with both envi- e m e rged (10, 11). Carri age of T N F Royal Hallamshire Hospital, Glossop Road, Sheffield S10 2JF, UK. ronmental and genetic fa c t o rs being +489A has been associated with both E-mail: [email protected] important with the latter contributing decreased risk of developing RA and a Received on March 7, 2002; accepted in around 30% of total susceptibility (1). 3 . 9 - fold reduced risk of deve l o p i n g revised form on June 4, 2002. The identifi c ation of suscep t i b i l i t y erosive RA in a Dutch study. Further- © Copyright CLINICAL AND determinants in a genetically complex more, this association was independent EXPERIMENTAL RHEUMATOLOGY 2002. disease such as RA is difficult, however of SE alleles (11). We have recently it is clear that a major genetic contribu- reported the association of TNF+ 489A Key words: Rheumatoid arthritis, tion, perhaps as much as one third of with DRB1*11 (12). We therefore un- TNF +489 polymorphism. the total, lies within the Major Histo- dertook to re-examine the role of the compatibility Complex (MHC). Alleles TNF+489 poly m o rphism in rheuma- of HLA-DRB1 which encode a similar toid arthritis by performing case con- sequence have been most convincingly trol studies in two European popula- implicated (2). This sequence, termed tions. the shared epitope (SE), encodes amino acids 69-73 of the mature peptide and Materials and methods is important in determining the reper- Patients and controls toire of peptides which are bound by Populations from two regions of Eur- the class II molecules and presented to ope we re used; from Galacia, S p a i n T cells (3). The linked DQ locus has ( C o n t ro l s , n = 145; pat i e n t s , n = 1 7 9 ) also been implicated but it is not clear and Sheffield, England (controls, n = if this association is primary or secon- 217; patients, n=238). All the Spanish dary to linkage disequilibrium with SE RA patients we re re c ruited from the alleles (4). Division of Rheumatology of the Hos- The class III region of the MHC lies pital Xeral-Calde (Lugo, Spain). This

829 BRIEF PAPER TNF+489 polymorphism and RA / A.S. Low et al. hospital is the only referral center for a tides and final concentrations (nM) as in the Spanish controls and pat i e n t s mixed rural (60%) and urban popula- follows: Forward: 5’-ATA CACACT respectively. tion of almost 250,000 people living in TAG TGA GCA CCT TCC AT- 3 ’ Carriage of allele 3 of the TNFb micro- the area surrounding Lugo city, in the (300), Reverse: 5’- GGT GAA AGA satellite had previously been reported m i ddle of the province of Lugo , i n TGT GCG CTG ATA G-3’ ( 3 0 0 ) , to be an independent risk factor for RA Galicia, Northwestern Spain. This long Allele 1 Probe (FAM label) 5’-CGT in this Spanish population (16). We time neglected area of the inner of CTT TCT CCA CGT TTT TTT CTC therefore examined the association of Galicia has important peculiarities pre- TCC AT- 3 ’ (50) and Allele 2 Pro b e TNF+489 alleles with TNF microsatel- viously reported elsewhere (13) (14). (TET label) 5’- CGT CTT TCT CCA lite alleles. Significant associat i o n s Of note, it is not a coastal area and it TGT TTT TTT CTC TCC AT C - 3 ’ were found in the Spanish control pop- has been relatively isolated from the (100) in a total volume of 25 µl con- ulation between TNF+489A and TNF- rest of Galicia for many centuries due taining 1x TaqMan Universal Master a10 (c2 = 28, p < 0.0001) and TNFd4 to geographical problems. This popula- Mixª and made up with sterile water. (c2 =4.8, p = 0.03). No association was tion seems to be Caucasoid of Celtic The cy cling conditions we re as fo l- found between TNF+489A and TNFb3 origin (15), is relatively static, and no low s : an initial cy cle of 50¡C for 2 (c2 = 2.2, p = 0.14) in the Spanish major migration has occurred in the m i nu t e s , 95¡C for 10 minu t e s , fo l- patient cohort. area during the last two decades. lowed by 40 cycles of 95¡C for 15 sec- Finally we examined if this TNF mark- The English cohorts consisted of ran- onds and 62¡C for 1 minute, with a er was associated with clinical features dom blood donor and patients attend- final holding cycle of 15¡C. Genotype of RA. Although the nu m b e rs of ing the Rheumatology outpatient clinic was ascertained using an ABI Prism¨ patients in some groups were relatively in the Royal Hallamshire Hospital, 7200 Sequence Detection System small, we did not find any evidence of S h e ffi e l d. All patients sat i s fied the machine. a genetic association of this marke r 1987 revised A m e rican College of with the presence of radiological ero- Rheumatology criteria for the diagno- Statistical analysis sions, subcutaneous nodules or rheum- sis of RA. Controls we re ethnically The strength of association betwe e n atoid factor in the Spanish population m at ch e d, h e a l t hy individuals. Mea- RA and SE or TNF +489 alleles was (Table II). sures of clinical phenotype including assessed using odds ratio (OR) and the presence of radiological erosions, 95% confidence intervals (CI). Th e Discussion r h e u m atoid nodules and rheumat o i d a s s o c i ations between TNF+489 and A s s o c i ation studies are a powe r f u l factor we re ava i l able in the Spanish TNF micro s atellites we re calculat e d method of detecting weak ge n e t i c population and have been reported pre- using Fishers Exact Test. The SE alle- effects in polygenic diseases however a viously (16). les were defined as DRB1* 01, *0401, major problem inherent to this type of *0404, *0405, *0408, *10. study is population stratification, it is HLA typing therefore essential that genetic associa- DNA was extracted from EDTA anti- Results tions are confirmed in separation popu- coagulated blood by standard methods. Strong associations with SE alleles of lations. In this study we were not able HLA-DRB1 was typed in the Spanish HLA-DRB1 were seen in both popula- to detect an association of TNF+489A p o p u l ation using a semi-automat e d tions; England OR = 2.9 (2.2-3.9), with the development of RA or with c o m m e rc i a l ly ava i l able reve rse dot Spain OR = 2.3 (1.6-3.3). Carriage of the presence of clinical marke rs of blot method, INNO-LIPA (Abbott lab- TNF+489 alleles did not differ be- severe disease.The original study from oratories, England) and, in the English tween controls and patients in either the Netherlands had reported a reduc- population, by polymerase chain reac- population (Table I). The frequency of tion in carri age of TNF+489A fro m tion sequence specific primer reactions the TNF+489A allele was identical 12% in controls to 6.8% and 7.1% in (PCR-SSP) amplification followed by (0.08) in the English controls and the patient populations. In add i t i o n agarose gel electrophoresis (17). patients compared with 0.13 and 0.09 carriage of TNF+489A was associated

TNF microsatellites TNF microsatellites were typed in the Table I. Carriage rates of TNF+489 alleles in two European control and RA populations. Spanish population and the re s u l t s TNF+489 TNF+489 Odds Ratio have been reported previously (16). G,G G,A or A,A

TNF +489 typing England Controls 181 35 Typing was performed by 5’-nuclease RA 201 37 1.1 (0.6-1.7) PCR using the PE Biosystems Taqman allelic discrimination system: 20 ng of Spain genomic DNA was used as template Controls 76 26 RA 112 24 1.6 (0.85-3.0) for amplifi c ation using oligo nu cl e o-

830 TNF+489 polymorphism and RA / A.S. Low et al. BRIEF PAPER

Table II. Carriage of the TNF+489A allele is not associated with the presence of radiologi- mune diseases may lie telomeric of the cal erosions, rheumatoid nodules or rheumatoid factor in Spanish patients. class II region. Erosions Nodules Rheumatoid factor Although we were not able to replicate the findings reported by van Krugten et TNF+489 - + - + - + al. implicating the TNF+489A allele G,G 30 69 99 13 28 84 with RA suscep t i b i l i t y, t h e re is an A,G 6 15* 21 3† 5 19¦ increasing body of evidence implicat- ing the telomeric region of the MHC in * † ¦ OR = 0.9 (0.3-2.6); OR = 1.1 (0.3-4.2); OR = 1.3 (0.4-3.7) the genetic back ground of RA. Th e identification of this locus should be with a 3.9-fold decrease in risk of Spanish RA families found preferential gre at ly fa c i l i t ated by our incre a s i n g developing erosive disease compared t ransmission of TNF haplotypes to knowledge of the gene content of the with that of patients who were homo- affected offspring which was indepen- MHC and the generation of a high den- zygous for the common allele (TNF+ dent from transmission of SE alleles sity genetic map of this region. 489G). These associations were found (18), these results were supported by a to be independent of SE alleles sug- study of 50 multiplex Irish RA families References 1. OLLIER W, WORTHINGTON J: Small fish in gesting that this marker might be an (19). Recently several case control stu- a big pond. Br J Rheumatol 1997; 36: 931- independent risk factor for the devel- dies, involving typing of a large num- 82. opment of rheumatoid arthritis. ber of polymorphic markers spanning 2. G R E G E R S E N P K , S I LVER J, W I N C H E S T E R H owever our study, i nvolving two the MHC, have also shown evidence RJ:The shared epitope hypothesis. An ap- p ro a ch to understanding the molecular l a rge populations from diffe rent re- for a second susceptibility locus in the genetics of susceptibility to rheumat o i d gions within Europe, did not find evi- region telomeric of DRB1 (20, 2 1 ) arthritis. Arthritis Rheum 1987; 30:1205-13. dence implicating carri age of T N F + with some evidence implicating a 70 3. PE N Z OT T I J E , NEPOM GT, LYBRAND T P: 489 in the genetic background of RA. kb region between the TNF and HLA- Use of T cell receptor/HLA-DRB1*04 mole- cular modeling to predict site-specific inter- With the combined data set of 692 B loci (22). This region is known to actions for the DR shared epitope associated observations we should be able to de- contain four ex p ressed genes; NFK- with rheumatoid art h ritis. A rt h ritis Rheum tect a difference in frequency of the BIL1 (IkB L ) , AT P 6 G, BAT1 and 1997; 40: 1316-26. TNF+489GA or +489AA genotypes of M I C B, wh i ch there fo re rep resent at- 4. DE VRIES N, VAN ELDERENC,TIJSSENH, VAN RIEL PL, VAN DE PUTTE LB: No support for 0.07 compared with the observed value tractive candidate genes. However, it HLA-DQ encoded susceptibility in rheuma- of 0.18 (these values chosen to match must be stressed that not all studies toid art h ritis [see comments]. A rt h ri t i s the 40% reduction reported previously have confirmed these associations to Rheum 1999; 42: 1621-7. (11), with 80% power at the 5% level be independent from DRB1 (23). 5. Complete sequence and gene map of a hu- man major histocompatibility complex. The of signifi c a n c e. No association be- Further evidence for the role of a sec- MHC sequencing consortium [see com- tween TNF +489 and SE +ve alleles ond genetic susceptibility locus within ments]. Nature 1999; 401: 921-3. was found.We have previously report- the MHC has also been rep o rted in 6. WILSON AG, SYMONS JA, McDOWELL TL, McDEVITT HO, DUFF GW: Effects of a poly- ed genetic association of TNF +489 A other autoimmune diseases. Two re- morphism in the human tumor necrosis fac- with HLA-DRB1*11 (12). The fre- p o rts have described associations of tor alpha promoter on transcriptional activi- quency of the TNF +489 allele seems carriage of TNF-308A with increased ty. Proc Nat Acad Sci (USA) 1997; 94:3195- to va ry across Europe from 0.12 in risk of developing systemic lupus ery- 9. 7. KNIGHT JC, UDALOVA I, HILL AVS, GREEN- I t a ly, Spain and the Netherlands to thematosus, independent from class II WOOD BM, PESHU N, MARSH K, 0.08 in England.The Spanish RA co- and complement cluster alleles (24, KW I AT K OWSKI D: A poly m o rphism that hort studied here has been previously 25). Recently other autoimmune dis- affects OCT-1 binding to the TNF promoter reported to show independent associa- eases including scleroderma (26), giant region is associated with seve re malari a . Nature Genetics 1999; 22: 145-50. tions with DRB1 and TNF alleles. This cell arteritis (27), coeliac disease (28) 8. WILSON AG, DE VRIES N, VANDE PUTTE LBA, may be due to linkage disequilibrium and myasthenia gravis (29) have also DUFF GW: A tumour necrosis factor alpha with a nearby va ri a n t , h oweve r, o u r been reported to show secondary inde- polymorphism is not associated with rheu- results are consistent with the TNF+ pendent genetic associations with the matoid arthritis. Ann Rheum Dis 1995; 54: 601-3. 489 variant being neutral in RA. telomeric class III/centromeric class I 9 BR I N K M A N B M N, H U I Z I N G A T W J, K U R- Despite these results, there is increas- regions. Susceptibility genes fo r BAN SS et al.: Tumour necrosis factor a gene ing evidence that a second susceptibili- autoimmune diseases have been shown polymorphisms in rheumatoid arthritis:asso- ty locus for RA lies in the distal class to cluster non-randomly across the ge- ciation with susceptibility to, or severity of, disease? Br J Rheumatol 1997; 36: 516-21. III or class I regions of the MHC. Two nome, suggesting that distinct autoim- 10. MULLIGHAN CG, FANNING GC, C H A P E L family based studies using the trans- mune diseases may be controlled by HM,WELSHKI: TNF and lymphotoxin-alpha mission disequilibrium test have exam- common sets of susceptibility ge n e s p o ly m o rphisms associated with common va ri able immu n o d e fi c i e n cy : Role in the ined if TNF microsatellite alleles are (30). The above reports are consistent p at h ogenesis of gra nu l o m atous disease. J more frequently transmitted to affected with the idea that a second MHC sus- Immunol 1997; 159: 6236-41. offspring than expected. A study of 52 ceptibility locus for a range of autoim- 11. VAN KRUGTEN MV, HUIZINGA TWJ, KAIJZEL

831 BRIEF PAPER TNF+489 polymorphism and RA / A.S. Low et al.

EL et al.: Association of the TNF+489 poly- PASCUAL-SALCEDO D, CONEJERO L,ALVES associated with systemic lupus erythemato- m o rphism with susceptibility and ra d i o- H , BALSA A , DE LA C O N C H A E G: P ri m a ry sus in African-Americans. Arthritis Rheum graphic damage in rheumatoid art h ri t i s . association of -region 1997; 40: 2207-11. Genes and Immunity 1999; 1: 1-6. genetic marke rs with susceptibility to 25. VAN ROOD MJ, VAN KRUGTEN MV, ZANELLI 12. LOW AS, AZMY I, SHARAF N , CANNINGS C, rheumatoid arthritis. Arthritis Rheum 2000; E et al.: TNF-308A and HLA-DR3 alleles WILSON AG: A s s o c i ation between two tu- 43: 1366-70. contribute independently to susceptibility to mour necrosis factor intronic polymorphisms 19. MULCAHY B,WALDRON-LYNCH F, McDER- systemic lupus ery t h e m atosus. A rt h ri t i s and HLA alleles. Eur J Immunogen 2002; 29: MOTT MF et al.: Genetic variability in the Rheum 2000; 43: 129-34. 31-4. tumor necrosis fa c t o r- ly m p h o t oxin regi o n 26. FRANK KH,FUSSEL M,CONRAD K,RIHS HP, 13. G O N Z A L E Z - G AY MA, G A R C I A - P O R RUA C: i n fluences susceptibility to rheumat o i d KOCH R, GEBHARDT B , MEHLHORN J: Dif- 1999. Systemic vasculitis in adults in North- arthritis. Am J Hum Genetics 1996; 59: 676- ferent distribution of HLA class II and tumor western Spain, 1988-1997: Clinical and epi- 83. n e c rosis factor alleles (TNF-308.2, T N Fa 2 demiologic aspects. Medicine 1999; 78:292- 20. Z A N E L L I E , J O N E S G, PA S C UAL M et al. : m i c ro s atellite) in anti-topoisomerase I re- 308. The telomeric part of the HLA region predis- sponders among scleroderma patients with 14. GO N Z A L E Z - G AY MA, G A R C I A - P O R RUA C, poses to rheumatoid arthritis independently and without exposure to quartz/metal dust. BRANAS F, LOPEZ-LAZARO L, OLIVIERI, I: of the class II loci. Hum Immunol 2001; 62: Arthritis Rheum 1998; 41: 1306-11. Epidemiologic and clinical aspects of Beh- 75-84. 27. MATTEY DL, HAJEER A H , DA BABNEH A , cet's disease in a defined area of Northwest- 21. CASTRO F, ACEVEDO E, CIUSANI E, ANGU- THOMSON W, GONZALEZ-GAY MA,GARCIA- ern Spain,1988-1997. J Rheumatol 2002; 27: LO JA , WOLLHEIM FA , S A N D B E R G - WO L L- PORRUA C,OLLIER WE: Association of giant 703-7. HEIM M:Tumour necrosis factor microsatel- cell art e ritis and poly mya l gia rheumat i c a 15. DA BABNEH A , G O N Z A L E Z - G AY MA, G A R- lites and HLA-DRB1*, HLA-DQA1*, and with different tumor necrosis factor microsa- C I A - P O R RUA C, HAJEER A , THOMSON W, HLA-DQB1* alleles in Pe ruvian pat i e n t s tellite polymorphisms. Arthritis Rheum 2000; OLLIER W: Giant cell arteritis and polymyal- with rheumatoid art h ritis. Ann Rheum Dis 43: 1749-55. gia rheumatica can be differentiated by dis- 2001; 60: 791-5. 28. MCMANUS R, WILSON AG, MANSFIELD J, tinct patterns of HLA class II association. J 22. OTA M, KATSUYAMAY, KIMURA A et al.: A WEIR DG, DUFF GW, KELLEHER D: TNF2, a Rheumatol 1998; 25: 2140-5. second susceptibility gene for deve l o p i n g polymorphism of the tumour necrosis-alpha 16. HA J E E R A H , DA BA B N E H A , MAKKI RF e t rheumatoid arthritis in the human MHC is gene promoter, is a component of the celiac al.: Different gene loci within the HLA-DR localized within a 70-kb interval telomeric of disease major histocompatibility complex and TNF regions are independently associat- the TNF genes in the HLA class III region. haplotype. Eur J Immunol 1996; 26: 2113-8. ed with susceptibility and severity in Spanish Genomics 2001; 71: 263-70. 29. JANER M, COWLAND A, PICARD J et al.: A rheumatoid arthritis patients. Tissue Antigens 23. HAJEER AH, WORTHINGTON J, SILMAN AJ , s u s c eptibility region for myasthenia grav i s 2002; 55: 319-25. OLLIER WE: Association of tumor necrosis extending into the HLA-class I sector telom- 17. BUNCE M, O'NEILL CM, BARNARDO MC et factor micro s atellite poly m o rphisms with eric to HLA-C. Human Immunology 1999; al.:Phototyping:comprehensive DNA typing HLA-DRB1*04-bearing haplotypes in rheu- 60: 909-17. for HLA-A, B, C , D R B 1 , D R B 3 , D R B 4 , m atoid art h ritis patients. A rt h ritis Rheum 30. BECKER KG, SIMON RM, BAILEY-WILSON JE DRB5 & DQB1 by PCR with 144 primer 1996; 39: 1109-14. et al.: Clustering of non-major histocompati- m i xes utilizing sequence-specific pri m e rs 24. SULLIVAN KE, WOOTEN C,SCHMECKPEPER bility complex susceptibility candidate loci in (PCR-SSP). Tissue An t i ge n s 1995; 46: 35 5 - 6 7 . B J, GOLDMAN D, P E T R I M A: A pro m o t e r human autoimmune diseases. Proc Nat Acad 18. MA RTINEZ A , F E R NA N D E Z - A R QU E RO M, p o ly m o rphism of tumor necrosis factor a Sci (USA) 1998; 95: 9979-84.

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