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Confirmation of TNIP1 but not RHOB and PSORS1C1 as systemic sclerosis risk factorsin a large independent replication study

Bossini-Castillo Lara Ezequiel Martin Jose Broen Jasper Simeon Carmen P BerettaLorenzo Gorlova Olga Y Vonk Madelon C Ortego-Centeno Norberto Espinosa GerardCarreira Patricia Garcia de la Pena Paloma Oreiro Natividad Andres Roman-Ivorra JoseJesus Castillo Maria Gonzalez-Gay Miguel A Saez-Comet Luis Castellvi IvanSchuerwegh Annemie J Voskuyl Alexandre E Hoffmann-Vold Anna-Maria HesselstrandRoger Nordin Annika Lunardi Claudio Scorza Raffaella van Laar Jacob M Shiels PaulG Herrick Ariane Worthington Jane Fonseca Carmen Denton Christopher Tan FilemonK Arnett Frank C Assassi Shervin Koeleman Bobby P Mayes Maureen D RadstakeTimothy R D J Martin JavierPublished inAnnals of the Rheumatic Diseases

DOI101136annrheumdis-2012-201888

2013

Link to publication

Citation for published version (APA)Bossini-Castillo L Ezequiel Martin J Broen J Simeon C P Beretta L Gorlova O Y Vonk M COrtego-Centeno N Espinosa G Carreira P Garcia de la Pena P Oreiro N Andres Roman-Ivorra JJesus Castillo M Gonzalez-Gay M A Saez-Comet L Castellvi I Schuerwegh A J Voskuyl A E Martin J (2013) Confirmation of TNIP1 but not RHOB and PSORS1C1 as systemic sclerosis risk factors in alarge independent replication study Annals of the Rheumatic Diseases 72(4) 602-607httpsdoiorg101136annrheumdis-2012-201888Total number of authors37

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EXTENDED REPORT

Confirmation of TNIP1 but not RHOB and PSORS1C1as systemic sclerosis risk factors in a largeindependent replication studyLara Bossini-Castillo1 Jose Ezequiel Martin1 Jasper Broen2 Carmen P Simeon3

Lorenzo Beretta4 Olga Y Gorlova5 Madelon C Vonk2 Norberto Ortego-Centeno6

Gerard Espinosa7 Patricia Carreira8 Paloma Garciacutea de la Pentildea9 Natividad Oreiro10

Joseacute Andreacutes Romaacuten-Ivorra11 Mariacutea Jesuacutes Castillo12 Miguel A Gonzaacutelez-Gay13

Luis Saacuteez-Comet14 Ivan Castellviacute15 Annemie J Schuerwegh16 Alexandre E Voskuyl17

Anna-Maria Hoffmann-Vold18 Roger Hesselstrand19 Annika Nordin20 Claudio Lunardi21

Raffaella Scorza4 Jacob M van Laar22 Paul G Shiels23 Ariane Herrick24

Jane Worthington24 Carmen Fonseca25 Christopher Denton25 Filemon K Tan26

Frank C Arnett26 Shervin Assassi26 Bobby P Koeleman27 Maureen D Mayes26

Timothy R D J Radstake228 Javier Martin1 the Spanish Scleroderma Group

Additional supplementarydata are published online onlyTo view these files please visitthe journal online (httpdxdoiorg101136annrheumdis-2012-201888bmjcom)

For numbered affiliations seeend of article

Correspondence toDr Lara Bossini-CastilloDepartment of ImmunologyInstituto de Parasitologiacutea yBiomedicina Loacutepez-NeyraIPBLN-CSIC Consejo Superiorde Investigaciones CientiacuteficasParque Tecnoloacutegico Cienciasde la Salud Avenida delConocimiento sn 18100-Armilla Granada Spainlarabcipbcsices

Received 18 April 2012Accepted 20 July 2012Published Online First15 August 2012

ABSTRACTIntroduction A recent genome-wide association studyin European systemic sclerosis (SSc) patients identifiedthree loci (PSORS1C1 TNIP1 and RHOB) as novelgenetic risk factors for the disease The aim of this studywas to replicate the previously mentioned findings in alarge multicentre independent SSc cohort of CaucasianancestryMethods 4389 SSc patients and 7611 healthy controlsfrom different European countries and the USA wereincluded in the study Six single nucleotidepolymorphisms (SNP) rs342070 rs13021401 (RHOB)rs2233287 rs4958881 rs3792783 (TNIP1) andrs3130573 (PSORS1C1) were analysed Overallsignificance was calculated by pooled analysis of all thecohorts Haplotype analyses and conditional logisticregression analyses were carried out to explore furtherthe genetic structure of the tested lociResults Pooled analyses of all the analysed SNPs inTNIP1 revealed significant association with the wholedisease (rs2233287 pMH=194times10minus4 OR 119rs4958881 pMH=326times10minus5 OR 119 rs3792783pMH=216times10minus4 OR 119) These associations weremaintained in all the subgroups considered PSORS1C1comparison showed association with the complete set ofpatients and all the subsets except for the anti-centromere-positive patients However the associationwas dependent on different HLA class II alleles Thevariants in the RHOB gene were not associated with SScor any of its subsetsConclusions These data confirmed the influence ofTNIP1 on an increased susceptibility to SSc and reinforcedthis locus as a common autoimmunity risk factor

INTRODUCTIONSystemic sclerosis or scleroderma (SSc) is acomplex autoimmune disorder that affects the con-nective tissue causing fibrosis in the skin and

different internal organs1 The contribution ofdifferent genetic factors to the development andprognosis of the disease is now widely accepted2

Over the past few years genome-wide associationstudies (GWAS) have been a useful tool in thegenetic dissection of autoimmune pathologies andother complex diseases3 Radstake et al4 performedthe first SSc GWAS in Caucasian populationswhich represented the first large-scale GWAS inSSc This work reinforced the association withinthe HLA region especially with the HLA-DQB1gene which was also reported in a comprehensivemultiethnic SSc HLA study5 It also confirmed theassociations found in STAT4 and IRF5 and identi-fied CD247 as a new SSc risk locus It is worthmentioning that the role of CD247 in SSc hasrecently been independently replicated6 ThisGWAS has led to three follow-up studies whichhave described several novel SSc susceptibilityfactors ie IRF8 GRB10 SOX5 NOTCH4IL12RB2 CSK PSD3 and NFKB17ndash9 InterestinglySOX5 and NOTCH4 are directly related to thefibrotic process which is a main hallmark of SSc

A GWAS has recently been performed in aFrench Caucasian SSc discovery cohort10 In thisGWAS 17 single-nucleotide polymorphisms (SNP)showing tier two associations were selected forfollow-up in independent cohorts Three of theselected SNP were located within the HLA regioncorresponding to the HLA-DQB1 and PSORS1C1genes and the remaining SNP were located in sixindependent non-HLA loci After the replicationstep the associations of HLA-DQB1 CD247 STAT4and IRF5 were confirmed and six SNP located inthree loci (TNIP1 RHOB PSORS1C1) were pro-posed as novel SSc risk factors

It has been observed that associations identifiedfrom a single GWAS even passing the establishedstatistical significance thresholds tend to have

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inflated effect sizes11 This effect size is called the winner rsquoscurse and it also affects the predictive ability of the discoveredassociations and the estimate of the risk variance explained bythe associations11 Replication in independent comparablepopulations is thus essential for firmly establishing a genotypendashphenotype association11 12 Therefore we aimed to perform alarge-scale replication study of the novel SSc genetic risk factorsidentified by GWAS strategy in an independent white Europeanand US SSc population

PATIENTS AND METHODSSubjects4389 SSc patients and 7611 controls of Caucasian ancestry(Spain The Netherlands USA Italy Sweden UK and Norway)were included in this study Patients were classified as havinglimited or diffuse SSc as defined by LeRoy et al13 The follow-ing clinical data were collected for ascertainment of the clinicalphenotype of the patients with SSc age gender disease dur-ation and presence of SSc-associated autoantibodies anti-topoisomerase (ATA) and anti-centromere (ACA)Supplementary table S1 (available online only) shows thecohort-specific SSc patient data The control population con-sisted of unrelated healthy individuals recruited in the samegeographical regions as SSc patients and matched by age sexand ethnicity with the SSc patient groups Local ethics com-mittees from all the participating centres approved the studyBoth patients and controls were included in the study afterwritten informed consent

In the meta-analysis with previously published data byAllanore et al10 which includes 2246 SSc patients and 5702healthy controls from France Italy Germany and EasternEurope the total cohort size reached 6635 patients and 13 313controls (except for rs13021401 and rs3792783 which were notavailable for The Netherlands and US GWAS cohortsrespectively)

GenotypingGenotype data of six SNP (rs342070 rs13021401 (RHOB)rs2233287 rs4958881 rs3792783 (TNIP1) and rs3130573(PSORS1C1)) was obtained from both available GWAS geno-typing platforms and SNP genotyping assays When possiblegenotypes from the Spanish Dutch and US cohorts fromRadstake et al4 were used (Spain I The Netherlands I and US Icohorts) In addition additional Spanish SSc patients and con-trols were genotyped using the Illumina HumanCytoSNP-12DNA Analysis BeadChip and Illumina Human1M-Duo DNAAnalysis BeadChip ((Illumina Inc San Diego CA USA)respectively this information was thus included in the Spain Iset when available The remaining European cohorts (Spain IIThe Netherlands II Italy Sweden UK and Norway) were ana-lysed using TaqMan SNP genotyping assays in a 7900HTReal-Time PCR System from Applied Biosystems following themanufacturer rsquos suggestions (Applied Biosystems Foster CityCalifornia USA) The differences in the number of samplesincluded in the analyses of each polymorphism correspond tothe availability of the genotype data in each platform (seesupplementary table S2 available online only) Stringentquality control filters and principal component analysis wereapplied to the GWAS and the HLA imputed data as describedin Radstake et al4 The genotyping call-rate for the individualsgenotyped using TaqMan assays reached rs342070 9377rs13021401 9498 rs2233287 9608 rs4958881 9375rs3792783 9329 and rs3130573 9519

Statistical analysisPLINK (V107) software (httppngumghharvardedupurcellplink) was used for individual population association tests(significance was calculated by 2times2 contingency tables andFisher rsquos exact test or χ2 when necessary and in the case of thehaplotypes each haplotype was tested against all others) logis-tic regression and conditional logistic regression analyses Thedifferent cohorts were considered covariables in the logisticregression analyses OR and their 95 CI were reported TNIP1haplotypes were constructed using PLINK (V107) andHaploView 42 (httpwwwbroadinstituteorghaploviewhaploview) only with those individuals successfully genotypedfor the three included variants (2432 SSc patients and 3496healthy controls) The BreslowndashDay test was performed asimplemented in PLINK and StatsDirect to assess the homogen-eity of the association among populations Pooled analyses andmeta-analyses were carried out using a MantelndashHaenszel testunder a fixed effects by PLINK (V107) METAL14 (httpwwwsphumicheducsgabecasismetal) and StatsDirect (V266StatsDirect Ltd) in the case of haplotypes Significant hetero-geneity among populations was found in the meta-analysis ofRHOB locus polymorphisms consequently in this case arandom effects model was applied using StatsDirect Genotypicfrequency distributions for the meta-analysis were kindly pro-vided by Allanore et al10 for the meta-analysis by personal com-munication All cohorts were in HardyndashWeinberg equilibriumat a significance level of 001 for all the included SNP Powerwas calculated using the software Power Calculator for GeneticStudies 2006 and assuming an additive model at the 5 signifi-cance level and previously reported OR (rs342070 minor allele(A) frequency (MAF) 0226 OR 120 rs13021401 MAF 0225OR 121 rs2233287 MAF 0096 OR 131 rs4958881 MAF0115 OR 129 rs3792783 MAF 0152 OR 129 rs3130573MAF 0321 OR 125)15

RESULTSNon-HLA loci analysisIn this study we analysed five SNP located in two non-HLAloci TNIP1 and RHOB Regarding the TNIP1 locus we repli-cated the previously described associations with rs4958881showing the most significant relationship pMH=326times10minus5OR 119 95 CI 109 to 129 (table 1) We also observed thatthe associations were consistent through the different clinicaland serological subsets The BreslowndashDay test showed no evi-dence of interpopulation heterogeneity either in the wholedisease analyses or in the stratified groups (supplementary tableS3 available online only shows individual cohort analyses)Statistical power was over 99 for the three SNP Moreover allthe TNIP1 genetic variants showed significant association atGWAS level in the meta-analysis with the initial report(rs2233287 pMH=17times10minus9 OR 123 95 CI 115 to 132rs4958881 pMH=288times10minus11 OR 124 95 CI 116 to 132rs3792783 pMH=911times10minus16 OR 131 95 CI 123 to 140figure 1)

As previously described the three SNP examined belong tothe same haplotype block10 As reported by Allanore et al10 thepolymorphisms studied in the TNIP1 region showed moderateto high linkage disequilibrium (see supplementary figures S1and S2 available online only) Therefore haplotype analysiswas performed The BreslowndashDay test show homogeneityin the association of the haplotypes among populationsHaplotype block analysis revealed the association of two haplo-types with the disease Haplotypes CTT and TCC (SNP order

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rs2233287ndashrs4958881ndashrs3792783) represent the combinationsof the major and minor alleles of each SNP respectively andsubsequently show a protective or a susceptibility role that isconcordant with the individual SNP associations ie majoralleles are protective while minor alleles are risk variants (seesupplementary table S4 available online only) However haplo-type block analysis did not show more significant p valuesthan individual SNP analyses and no additive or multiplicativeeffect of the SNP was observed With the aim of clarifying pos-sible underlying dependence among the SNP we performedconditional logistic regression analysis Nevertheless due to thelinkage disequilibrium between the analysed SNP this approachdid not enable us to identify an independent association signal(see supplementary table S5 available online only)

As shown in table 1 none of the tested polymorphisms inRHOB showed significant associations with SSc or any of theexamined subgroups Only weak association signals could bedetected in the Italian cohort The power for the analyses ofthe rs342070 and rs13021401 RHOB genetic variants in theoverall cohort was of 99 in both cases Meta-analyses withthe previous report showed significant OR heterogeneity in theBreslowndashDay tests and no significant association under arandom effects model for both polymorphisms (rs342070Prandom=019 rs13021401 Prandom=013)

PSORS1C1 analysisThe study of the PSORS1C1 reported variant rs3130573showed a suggestive but heterogeneous association of this poly-morphism with increased SSc susceptibility (table 2) Moreoverthe association was maintained in all the subgroups (includingthe ACA-negative and ATA-negative subsets) except for theACA-positive patients Considering that the association of theHLA region with SSc is influenced primary by the autoanti-body profile of the patients5 7 we aimed to test for an uncov-ered influence of the HLA genes We thus carried out astep-wise logistic regression conditional analysis of the analysedPSORS1C1 variant with all the independent signals from themost significant to the lowest observed p values in the HLAregion The considered conditions included SNPs imputed ami-noacidic positions and imputed classic HLA-alleles (as describedin Raychaudhuri et al)16 in the analysed GWAS cohorts(Spain I The Netherlands I and USA I) (unpublished data)Standard logistic regression analyses in the GWAS cohortsshowed evidence of association only in the whole diseaseversus controls and in the ACA-negative patients versus con-trols comparisons (Plog=0034 OR 109 Plog=001 OR 112respectively) However the previously mentioned associationwith the whole set of SSc patients lost its significance whenit was conditioned to the HLA-DPB11301 allele (p value

Table 1 Pooled analysis of the novel SSc non-HLA susceptibility loci

Genotype N () Allele test

CHR BP SNP Locus 12 Subgroup (N) 11 12 22 MAF N () pMH OR (95 CI) pBD

2 20548952 rs342070 RHOB CT Controls (n=7193) 448 (623) 2713 (3772) 4032 (5605) 3609 (2509)SSc (n=4249) 292 (687) 1515 (3566) 2442 (5747) 2099 (2470) 076 101 (095 to 108) 008lcSSc (n=2649) 184 (695) 953 (3598) 1512 (5708) 1321 (2493) 075 101 (094 to 109) 024dcSSc (n=1227) 80 (652) 444 (3619) 703 (5729) 604 (2461) 095 100 (090 to 110) 021ACA+ (n=1492) 92 (617) 529 (3546) 871 (5838) 713 (2389) 052 097 (088 to 107) 052ATA+ (n=864) 71 (822) 303 (3507) 490 (5671) 445 (2575) 022 108 (096 to 121) 004

2 20552000 rs13021401 RHOB TC Controls (n=6557) 405 (618) 2416 (3685) 3736 (5698) 3226 (2460)SSc (n=4259) 280 (657) 1524 (3578) 2455 (5764) 2084 (2447) 061 102 (095 to 109) 006lcSSc (n=2644) 174 (658) 966 (3654) 1504 (5688) 1314 (2485) 049 103 (095 to 111) 011dcSSc (n=1239) 79 (638) 444 (3584) 716 (5779) 602 (2429) 096 100 (090 to 110) 038ACA+ (n=1487) 91 (612) 530 (3564) 866 (5824) 712 (2394) 091 099 (090 to 109) 039ATA+ (n=872) 65 (745) 316 (3624) 491 (5631) 446 (2557) 017 109 (097 to 122) 011

5 150420290 rs2233287 TNIP1 TC Controls (n=7164) 93 (130) 1226 (1711) 5845 (8159) 1412 (985)SSc (n=4152) 52 (125) 859 (2069) 3241 (7806) 963 (1160) 194E-04 119 (108 to 130) 072lcSSc (n=2586) 33 (128) 528 (2042) 2025 (7831) 594 (1148) 169E-03 118 (107 to 131) 070dcSSc (n=1194) 15 (126) 253 (2119) 926 (7755) 283 (1185) 281E-03 123 (107 to 141) 076ACA+ (n=1442) 19 (132) 309 (2143) 1114 (7725) 347 (1203) 227E-03 122 (107 to 139) 086ATA+ (n=849) 14 (165) 186 (2191) 649 (7644) 214 (1260) 494E-03 126 (107 to 147) 073

5 150430429 rs4958881 TNIP1 CT Controls (n=7182) 131 (182) 1512 (2105) 5539 (7712) 1774 (1235)SSc (n=4226) 98 (232) 1035 (2449) 3093 (7319) 1231 (1456) 326E-05 119 (109 to 129) 033lcSSc (n=2637) 57 (216) 637 (2416) 1943 (7368) 751 (1424) 123E-03 117 (106 to 128) 058dcSSc (n=1213) 31 (256) 302 (2490) 880 (7255) 364 (1500) 557E-04 124 (110 to 141) 029ACA+ (n=1490) 31 (208) 378 (2537) 1081 (7255) 440 (1477) 329E-03 119 (106 to 134) 014ATA+ (n=868) 29 (334) 232 (2673) 607 (6993) 290 (1671) 131E-05 136 (119 to 157) 057

5 150435925 rs3792783 TNIP1 CT Controls (n=3704) 113 (305) 995 (2686) 2596 (7009) 1221 (1648)SSc (n=2704) 108 (399) 829 (3066) 1767 (6535) 1045 (1932) 216E-04 119 (109 to 131) 070lcSSc (n=1708) 65 (381) 518 (3033) 1125 (6587) 648 (1897) 444E-03 117 (105 to 130) 056dcSSc (n=715) 30 (420) 222 (3105) 463 (6476) 282 (1972) 541E-03 123 (106 to 142) 059ACA+ (n=1041) 40 (384) 326 (3132) 675 (6484) 406 (1950) 609E-03 120 (105 to 136) 018ATA+ (n=623) 24 (385) 208 (3339) 391 (6276) 256 (2055) 336E-03 126 (108 to 147) 070

Controls are used as reference for all comparisonsAll p values have been calculated for the allelic modelACA+ anti-centromere autoantibody-positive patients ATA+ anti-topoisomerase autoantibody-positive patients BP base pair CHR chromosome CTRL healthy controlsdcSSc diffuse cutaneous systemic sclerosis lcSSc limited cutaneous systemic sclerosis MAF minor allele (A) frequency pMH MantelndashHaenszel test under fixed effect pBDhomogeneity BreslowndashDay test 12 minor allelemajor allele SNP single nucleotide polymorphism

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Figure 1 (A) Forest plot for the meta-analysis of the rs2233287 TNIP1 genetic variant (B) Forest plot for the meta-analysis of the rs4958881 TNIP1genetic variant (C) Forest plot for the meta-analysis of the rs3792783 TNIP1 genetic variant

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conditioning on DPB113 01=006 OR conditioning onDPB113 01=108) HLA-DRB111 04 (p value conditioningon DRB111 04=008 OR conditioning on DRB111 04=107) and the HLA-DQA105 01 (p value conditioning onDQA105 01=036 OR conditioning on DQA105 01=104)alleles Moreover the association with the ACA-negative subsetof patients was also shown to be dependent on the HLA-DQA105 01 allele (p value conditioning on DQA105 01=027 OR add to DQA105 01=105) Therefore our datasuggest that the association of PSORS1C1 with SSc is not inde-pendent from the HLA region

DISCUSSIONIn this study we conducted a large multicentre replication ofthe novel SSc risk variants identified by Allanore et al10 andwe confirmed the association of the TNIP1 locus with SScHowever the associations observed in the RHOB gene and theindependence of PSORS1C1 from the HLA region were not sup-ported by our data

Due to the lack of association observed in the RHOB locuswe suggest that the initial association reported in this genemight have been a false positive finding It is worth mentioningthat RHOB has never been robustly associated with an auto-immune disease and the previously reported association withSSc in this gene did not reach the GWAS significance level evenafter replication10

PSORS1C1 was proposed as an HLA-independent SSc riskfactor10 The authors performed a dependence analysis control-ling for the described association in the HLA-DQB1 gene andreported the independence of both loci Nevertheless HLA-DQB1has been specifically related with the ACA-positive subset ofpatients5 7 and both in our data and in the previous study noassociation of PSORS1C1 with ACA positivity has beenshown10 Therefore a deeper analysis of this locus was neededand we performed for the first time conditional logistic regressionincluding all the independent signals in the HLA region In ourinitial approach we found a signal in the PSORS1C1 gene thatwas comparable to the one described in the previous work10

however a comprehensive analysis showed that the PSORS1C1association is dependent from the HLA-DPB113 01HLA-DQA105 01 and HLA-DRB111 04 alleles (especially theHLA-DQA10501) These HLA loci have previously beendescribed as ATA positivity risk factors5 7 which is consistentwith the lack of association in the ACA-positive subgroup

Altogether our data do not confirm PSORS1C1 as an independ-ent player in the SSc genetic susceptibility network

Regarding the association of TNFAIP3 interacting protein 1(TNIP1) our data clearly support TNIP1 as a SSc risk factorOur replication study confirmed that the association of thethree SNPs tested is maintained in all the subsets indicatingthat this association peak corresponds to the whole diseaseTherefore TNIP1 might be implicated in the development ofthe disease but may not act as a disease modifier RemarkablyTNIP1 is involved in HIV replication acts as a negative regula-tor of the nuclear factor κB pathway (a key regulator ofthe immune response which has also recently been associatedwith SSc)9 and also represses agonist-bound retinoic acidreceptors and peroxisome proliferator-activated receptors17ndash19

Furthermore recent studies focused on the control of TNIP1transcription have reported a complex mechanism behindTNIP1 expression that combines constitutive transcriptionfactors and inducible factors (nuclear factor κB and peroxisomeproliferator-activated receptors)20 Interestingly Allanore et al10

showed that the transcription and expression of TNIP1 isdecreased both in the skin of SSc patients and SSc culturedfibroblasts thus the anti-inflammatory effect of this moleculemay be reduced in SSc patients providing evidence for arelevant role of TNIP1 in the disease TNIP1 is also a well-established risk factor for different autoimmune diseases suchas psoriasis psoriatic arthritis and systemic lupus erythemato-sus21ndash23 Furthermore TNIP1 association with psoriasis has alsobeen reported in Asian populations24 suggesting that the roleof TNIP1 in autoimmune diseases is consistent through differ-ent ethnicities Therefore this locus can be considered acommon autoimmune disease risk factor that can be used as anew therapeutic target

To conclude our replication study has reinforced the influ-ence of TNIP1 in an increased susceptibility to SSc and its roleas a new player in the autoimmunity genetic backgroundFuture research will identify the causal variant for the TNIP1association and its implication in SSc pathophysiology

Author affiliations1Department of Immunology Instituto de Parasitologiacutea y Biomedicina Loacutepez-NeyraIPBLN-CSIC Granada Spain2Department of Rheumatology Radboud University Nijmegen Medical CentreNijmegen The Netherlands3Servicio de Medicina Interna Hospital Valle de Hebron Barcelona Spain4Department of Clinical Immunology and Rheumatology Allergy IRCCS FondazionePoliclinico-Mangiagalli-Regina Elena and University of Milan Milan Italy

Table 2 Pooled-analysis of the rs3130573 PSORS1C1 HLA-region genetic variant

Genotype N () Allele test

CHR BP SNP 12 Subgroup (N) 11 12 22 MAF N () pMH OR (95 CI) pBD

6 31214247 rs3130573 GA Controls (n=7139) 892 (1249) 3220 (4510) 3027 (4240) 5004 (3505)SSc (n=4130) 574 (1390) 1953 (4729) 1603 (3881) 3101 (3754) 117E-05 114 (107 to 121) 002lcSSc (n=2575) 350 (1359) 1221 (4742) 1004 (3899) 1921 (3730) 116E-03 112 (105 to 120) 007dcSSc (n=1187) 177 (1491) 561 (4726) 449 (3783) 915 (3854) 309E-04 118 (108 to 129) 007ACA+ (n=1446) 181 (1252) 666 (4606) 599 (4142) 1028 (3555) 028 105 (096 to 114) 012ACAminus (n=2511) 356 (1507) 1143 (4837) 864 (3656) 1855 (3925) 101E-07 121 (113 to 129) 005ATA+ (n=845) 123 (1456) 411 (4864) 311 (3680) 657 (3888) 648E-03 116 (104 to 129) 051ATAminus (n=3147) 413 (1392) 1395 (4700) 1160 (3908) 2221 (3742) 103E-04 113 (107 to 121) 003

Controls are used as reference for all comparisonsAll p values have been calculated for the allelic modelACAplusmn anti-centromere autoantibody-positivenegative patients ATAplusmn anti-topoisomerase autoantibody-positivenegative patients BP base pair CHR chromosome CTRLhealthy controls dcSSc diffuse cutaneous systemic sclerosis lcSSc limited cutaneous systemic sclerosis MAF minor allele (A) frequency pMH MantelndashHaenszel test underfixed effect pBD homogeneity BreslowndashDay test 12 minor allelemajor allele SNP single nucleotide polymorphism

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5Department of Epidemiology MD Anderson Cancer Center The University of TexasHouston Texas USA6Servicio de Medicina Interna Hospital Cliacutenico Universitario Granada Spain7Servicio de Enfermedades Autoinmunes Hospital Clinic Barcelona Spain8Servicio de Reumatologiacutea Hospital Universitario 12 de Octubre Madrid Spain9Servicio de Reumatologiacutea Hospital Madrid Norte Sanchinarro Madrid Spain10Servicio de Reumatologiacutea INIBIC-Hospital Universitario A Coruntildea La Coruntildea Spain11Servicio de Reumatologiacutea Hospital Universitari i Politecnic La Fe Valencia Spain12Servicio de Medicina Interna Hospital Virgen del Rociacuteo Sevilla Spain13Servicio de Reumatologiacutea Hospital Universitario Marqueacutes de Valdecilla IFIMAVSantander Spain14Unidad de Enfermedades Autoinmunes Sisteacutemicas Servicio de Medicina InternaHospital Universitario Miguel Servet Zaragoza Spain15Servicio de Reumatologiacutea Hospital Sant Pau Barcelona Spain16Department of Rheumatology Leiden University Medical Center LeidenThe Netherlands17Department of Rheumatology VU University Medical Center AmsterdamThe Netherlands18Department of Rheumatology Rikshospitalet Oslo University Hospital Oslo Norway19Department of Rheumatology Lund University Lund Sweden20Karolinska Institute Stockholm Sweden21Department of Medicine Universitagrave degli Studi di Verona Verona Italy22Musculoskeletal Research Group Institute of Cellular MedicineNewcastle University Newcastle UK23University of Glasgow Glasgow UK24Arthritis Research UK Epidemiology Unit The University of ManchesterManchester Academic Health Science Centre Manchester UK25Centre for Rheumatology Royal Free and University College Medical SchoolLondon UK26Department of Rheumatology and Clinical Immunogenetics The University of TexasHealth Science Center-Houston Houston Texas USA27Section Complex Genetics Department of Medical Genetics University MedicalCenter Utrecht Utrecht The Netherlands28Department of Rheumatology and Clinical Immunology Utrecht Medical CenterUtrech The NetherlandsFor members of the Spanish Scleroderma group see supplementary appendixavailable online only)

Acknowledgements The authors would like to thank Sofia Vargas Sonia Garciacuteaand Gema Robledo for excellent technical assistance and all the patients andcontrol donors for their essential collaboration They thank Banco Nacional de ADN(University of Salamanca Spain) and the Norwegian Bone Marrow Donor Registrywho supplied part of the control DNA samples They are also grateful to EUSTAR(the EULAR Scleroderma Trials and Research group) for the facilitation of thisproject

Contributors LBC and JEM contributed to the analysis and interpretation of dataand in drafting the article JB CPS LB OG MCV NOC GE PC PGdlP NO JARIMJC MAGG LSC IC AJS AEV AMHV RH AN CL RS JMvL PS AH JW CF CDFKT FCA and SA participated in analysis and interpretation of data and criticallyrevised the manuscript draft BPK MDM TRDJR and JM were involved in theconception and design of the study and critically revised the submitted version of themanuscript All authors approved the final version to be published

Funding This work was supported by the following grants JM was funded byGEN-FER from the Spanish Society of Rheumatology SAF2009ndash11110 from theSpanish Ministry of Science CTS-4977 from Junta de Andaluciacutea Spain in part byRedes Temaacuteticas de Investigacioacuten Cooperativa Sanitaria Programme RD080075(RIER) from Instituto de Salud Carlos III (ISCIII) Spain and by Fondo Europeo deDesarrollo Regional (FEDER) TRDJR was funded by the VIDI laureate from the DutchAssociation of Research (NWO) and Dutch Arthritis Foundation (NationalReumafonds) JM and TRDJR were sponsored by the Orphan Disease Programmegrant from the European League Against Rheumatism (EULAR) BPCK is supported bythe Dutch Diabetes Research Foundation (grant 200840001) and the Dutch ArthritisFoundation (Reumafonds grant NR 09-1-408) TW was granted by DFG WI 103161This study was also funded by PI-0590-2010 Consejeriacutea de Salud Junta deAndaluciacutea Spain The USA studies were supported by NIHNIAMS SclerodermaRegistry and DNA Repository (N01-AR-0-2251) NIHNIAMS-RO1-AR055258 andNIHNIAMS Center of Research Translation in Scleroderma (1P50AR054144) and theDepartment of Defense Congressionally Directed Medical Research Programmes(W81XWH-07-01-0111)

Competing interests None

Patient consent Obtained

Ethics approval Local ethics committees from all the participating centres approvedthe study

Provenance and peer review Not commissioned externally peer reviewed

REFERENCES1 Gabrielli A Avvedimento EV Krieg T Scleroderma N Engl J Med

20093601989ndash20032 Martin JE Bossini-Castillo L Martin J Unraveling the genetic component of

systemic sclerosis Hum Genet 20121311023ndash373 Visscher PM Brown MA McCarthy MI et al Five years of GWAS discovery Am J

Hum Genet 2012907ndash244 Radstake TR Gorlova O Rueda B et al Genome-wide association study of

systemic sclerosis identifies CD247 as a new susceptibility locus Nat Genet201042426ndash9

5 Arnett FC Gourh P Shete S et al Major histocompatibility complex (MHC) class IIalleles haplotypes and epitopes which confer susceptibility or protection insystemic sclerosis analyses in 1300 Caucasian African-American and Hispaniccases and 1000 controls Ann Rheum Dis 201069822ndash7

6 Dieude P Boileau C Guedj M et al Independent replication establishes the CD247gene as a genetic systemic sclerosis susceptibility factor Ann Rheum Dis2011701695ndash6

7 Gorlova O Martin JE Rueda B et al Identification of novel genetic markersassociated with clinical phenotypes of systemic sclerosis through a genome-wideassociation strategy PLoS Genet 20117e1002178

8 Bossini-Castillo L Martin JE Broen J et al A GWAS follow-up study reveals theassociation of the IL12RB2 gene with systemic sclerosis in Caucasian populationsHum Mol Genet 201221926ndash33

9 Martin JE Broen JC Carmona FD et al Identification of CSK as a systemicsclerosis genetic risk factor through genome wide association study follow-up HumMol Genet 2012212825ndash35

10 Allanore Y Saad M Dieude P et al Genome-wide scan identifies TNIP1PSORS1C1 and RHOB as novel risk loci for systemic sclerosis PLoS Genet20117e1002091

11 Ioannidis JP Thomas G Daly MJ Validating augmenting and refininggenome-wide association signals Nat Rev Genet 200910318ndash29

12 Chanock SJ Manolio T Boehnke M et al Replicating genotypendashphenotypeassociations Nature 2007447655ndash60

13 LeRoy EC Black C Fleischmajer R et al Scleroderma (systemic sclerosis)classification subsets and pathogenesis J Rheumatol 198815202ndash5

14 Willer CJ Li Y Abecasis GR METAL fast and efficient meta-analysis ofgenomewide association scans Bioinformatics 2010262190ndash1

15 Skol AD Scott LJ Abecasis GR et al Joint analysis is more efficient thanreplication-based analysis for two-stage genome-wide association studies NatGenet 200638209ndash13

16 Raychaudhuri S Sandor C Stahl EA et al Five amino acids in three HLA proteinsexplain most of the association between MHC and seropositive rheumatoid arthritisNat Genet 201244291ndash6

17 Gupta K Ott D Hope TJ et al A human nuclear shuttling protein that interactswith human immunodeficiency virus type 1 matrix is packaged into virions J Virol20007411811ndash24

18 Mauro C Pacifico F Lavorgna A et al ABIN-1 binds to NEMOIKKgamma andco-operates with A20 in inhibiting NF-kappaB J Biol Chem 200628118482ndash8

19 Gurevich I Aneskievich BJ Liganded RARalpha and RARgamma interact with butare repressed by TNIP1 Biochem Biophys Res Commun 2009389409ndash14

20 Gurevich I Zhang C Encarnacao PC et al PPARgamma and NF-kappaB regulatethe gene promoter activity of their shared repressor TNIP1 Biochim Biophys Acta201218191ndash15

21 Nair RP Duffin KC Helms C et al Genome-wide scan reveals association ofpsoriasis with IL-23 and NF-kappaB pathways Nat Genet 200941199ndash204

22 Bowes J Orozco G Flynn E et al Confirmation of TNIP1 and IL23A assusceptibility loci for psoriatic arthritis Ann Rheum Dis 2011701641ndash4

23 Gateva V Sandling JK Hom G et al A large-scale replication study identifiesTNIP1 PRDM1 JAZF1 UHRF1BP1 and IL10 as risk loci for systemic lupuserythematosus Nat Genet 2009411228ndash33

24 Sun LD Cheng H Wang ZX et al Association analyses identify six new psoriasissusceptibility loci in the Chinese population Nat Genet 2010421005ndash9

Ann Rheum Dis 201372602ndash607 doi101136annrheumdis-2012-201888 607

Basic and translational research

groupbmjcom on September 18 2013 - Published by ardbmjcomDownloaded from

doi 101136annrheumdis-2012-20188815 2012

2013 72 602-607 originally published online AugustAnn Rheum Dis Lara Bossini-Castillo Jose Ezequiel Martin Jasper Broen et al studyfactors in a large independent replication

as systemic sclerosis riskPSORS1C1 and RHOB but not TNIP1Confirmation of

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