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Trans-Ancestral Studies Fine Map the SLE-Susceptibility Locus TNFSF4 Harinder Manku1, Carl D. Langefeld2, Sandra G. Guerra3, Talat H. Malik4, Marta Alarcon-Riquelme5, Juan-Manuel Anaya6, Sang-Cheol Bae7, Susan A. Boackle8, Elizabeth E. Brown9, Lindsey A. Criswell10, Barry I. Freedman11, Patrick M. Gaffney12, Peter A. Gregersen13, Joel M. Guthridge12, Sang-Hoon Han7, John B. Harley14, Chaim O. Jacob15, Judith A. James12,16, Diane L. Kamen17, Kenneth M. Kaufman14, Jennifer A. Kelly12, Javier Martin18, Joan T. Merrill19, Kathy L. Moser12, Timothy B. Niewold20, So-Yeon Park7, Bernardo A. Pons-Estel21, Amr H. Sawalha22, R. Hal Scofield12,16, Nan Shen23, Anne M. Stevens24, Celi Sun12, Gary S. Gilkeson25, Jeff C. Edberg25, Robert P. Kimberly26, Swapan K. Nath12, Betty P. Tsao27, Tim J. Vyse1* 1Department of Medical & Molecular Genetics, King’s College London School of Medicine, Guy’s Hospital, London, United Kingdom, 2Wake Forest School of Medicine,Winston-Salem,NorthCarolina,UnitedStatesofAmerica,3CentreforRheumatology&ConnectiveTissueDiseases,RoyalFree&UniversityCollegeMedical School,London,UnitedKingdom,4DivisionofImmunologyandInflammation,ImperialCollege,London,UnitedKingdom,5CentroPfizer-UniversidaddeGranada- Junta de Andaluc´ıa de Geno´mica e Investigaciones Oncolo´gicas, Granada, Spain, 6Center for Autoimmune Diseases Research, Universidad del Rosario, Bogota, Colombia,7HospitalforRheumaticDiseases,HanyangUniversity,Seoul,SouthKorea,8DivisionofRheumatology,UniversityofColoradoDenver,Aurora,Colorado, UnitedStatesofAmerica,9DepartmentofEpidemiology,UniversityofAlabamaatBirmingham,Birmingham,Alabama,UnitedStatesofAmerica,10RosalindRussell MedicalResearchCenterforArthritis,UniversityofCaliforniaSanFrancisco,SanFrancisco,California,UnitedStatesofAmerica,11DepartmentofInternalMedicine, WakeForestSchoolofMedicine,Winston-Salem,NorthCarolina,UnitedStatesofAmerica,12ArthritisandClinicalImmunologyResearchProgram,OklahomaMedical ResearchFoundation,OklahomaCity,Oklahoma,UnitedStatesofAmerica,13TheRobertS.BoasCenterforGenomicsandHumanGenetics,FeinsteinInstitutefor MedicalResearch,NorthShoreLIJHealthSystem,Manhasset,NewYork,UnitedStatesofAmerica,14DivisionofRheumatology,CincinnatiChildren’sHospitalMedical Centre,Cincinnati,Ohio,UnitedStatesofAmerica,15TheLupusGeneticsGroup,DepartmentofMedicine,UniversityofSouthernCalifornia,LosAngeles,California, United States of America, 16Department of Medicine, University of Oklahoma Healthy Sciences Center, Oklahoma City, Oklahoma, United States of America, 17DivisionofRheumatology,MedicalUniversityofSouthCarolina,Charleston,SouthCarolina,UnitedStatesofAmerica,18InstitutodeParasitologiayBiomedicina Lopez-Neyra,ConsejoSuperiordeInvestigacionesCientificas,Granada,Spain,19ClinicalPharmacology,OklahomaMedicalResearchFoundation,OklahomaCity, Oklahoma,UnitedStatesofAmerica,20DivisionsofRheumatologyandImmunology,MayoClinic,Rochester,Minnesota,UnitedStatesofAmerica,21Sanatorio Parque,Rosario,Argentina,22DivisionofRheumatology,DepartmentofInternalMedicine,UniversityofMichigan,AnnArbor,Michigan,UnitedStatesofAmerica, 23Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai, China, 24Center for Immunity and Immunotherapies, Seattle Children’s ResearchInstitute,Seattle,Washington,UnitedStatesofAmerica,25DivisionofRheumatologyandImmunology,MedicalUniversityofSouthCarolina,Charleston, SouthCarolina,UnitedStatesofAmerica,26DivisionofClinicalImmunologyandRheumatology,DepartmentofMedicine,UniversityofAlabamaatBirmingham, Birmingham,Alabama,UnitedStatesofAmerica,27DivisionofRheumatology,DepartmentofMedicine,DavidGeffenSchoolofMedicineatUCLA,LosAngeles, California,UnitedStatesofAmerica Abstract Wepreviouslyestablishedan80 kbhaplotypeupstreamofTNFSF4asasusceptibilitylocusintheautoimmunediseaseSLE. SLE-associated alleles at this locus are associated with inflammatory disorders, including atherosclerosis and ischaemic stroke.InEuropeans,the TNFSF4causalvariantshaveremained elusiveduetostronglinkagedisequilibriumexhibitedby allelesspanningtheregion.Usingatrans-ancestralapproachtofine-mapthelocus,utilising17,900SLEandcontrolsubjects includingAmerindian/Hispanics(1348cases,717controls),African-Americans(AA)(1529,2048)andbetterpoweredcohorts of Europeans and East Asians, we find strong association of risk alleles in all ethnicities; the AA association replicates in African-American Gullah (152,122). The best evidence of association comes from two adjacent markers: rs2205960-T (P=1.71610234, OR=1.43[1.26–1.60]) and rs1234317-T (P=1.16610228, OR=1.38[1.24–1.54]). Inference of fine-scale recombinationratesforallpopulationstestedfindsthe80kbriskandnon-riskhaplotypesinallexceptAfrican-Americans. Inthis populationthedecayofrecombination equatestoan11kbrisk haplotype,anchoredinthe 59regionproximal to TNFSF4andtaggedbyrs2205960-Tafter1000Genomesphase1(v3)imputation.Conditionalregressionanalysesdelineate the59risksignaltors2205960-Tandtheindependentnon-risksignaltors1234314-C.Ourcase-onlyandSLE-controlcohorts demonstraterobustassociationofrs2205960-Twithautoantibodyproduction.Thers2205960-Tispredictedtoformpartofa decamericmotifwhichbindsNF-kBp65withincreasedaffinitycomparedtors2205960-G.ChIP-seqdataalsoindicateNF-kB interactionwiththeDNAsequenceatthispositioninLCLcells.Ourresearchsuggestsassociationofrs2205960-TwithSLE across multiple groups and an independent non-risk signal at rs1234314-C. rs2205960-T is associated with autoantibody productionandlymphopenia.OurdataconfirmaglobalsignalatTNFSF4andarolefortheexpressedproductatmultiple stages of lymphocyte dysregulation during SLE pathogenesis. We confirm the validity of trans-ancestral mapping in a complex trait. PLOSGenetics | www.plosgenetics.org 1 July2013 | Volume 9 | Issue 7 | e1003554 TransAncestralFineMappingofSLELocusTNFSF4 Citation: Manku H, Langefeld CD, Guerra SG, Malik TH, Alarcon-Riquelme M, et al. (2013) Trans-Ancestral Studies Fine Map the SLE-Susceptibility Locus TNFSF4.PLoSGenet9(7):e1003554.doi:10.1371/journal.pgen.1003554 Editor:MarkI.McCarthy,UniversityofOxford,UnitedKingdom ReceivedOctober22,2012;AcceptedApril23,2013;PublishedJuly18,2013 Copyright: (cid:2)2013 Manku et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding:ThisworkwasmadepossiblethroughtheAllianceforLupusResearch(ALR)GenesfromSLEGEN:TheLupusGeneticsConsortium.Additionalsupport forthisworkwasobtainedfromWellcomeTrustprogrammegrant085492(TJV,HM),WellcomeTrustprojectgrant083167(TJV,HM),PO1AR49084(RPKandEEB), R01AR33062(RPK),R01AR62277(JBHandKLM),R01AR043274(KLM),R01AI063274(PMG),R37AI24717(JBH),R01AR042460(JBH),P01AI083194(JBH),P20 RR020143(JBH),P30AR055385(EEB),K08AI083790(TBN),LRPAI071651(TBN),R01CA141700(MAR),RC1AR058621(MAR),UL1RR024999(TBN),R01AR051545 (AMS),P30AR053483(JAJandJMG),U19AI082714(JAJandJMG),P30GM103510(JAJandJMG)andU01AI101934(JAJ)R21AI070304(SAB),R01AR43814(BPT), P60AR053308(LAC),M01RR-00079(LAC),R01AR057172(COJ),P60-AR049459(GSGandDLK),UL1-RR029882(GSGandDLK)andCTSAGrantNumberIULI RR025014-02(AMS),AI094377(SKN),AR060366(SKN).ThisstudywasalsosupportedbytheArthritisResearchUK(SGG,TJV),LupusResearchInstitute(TBN,BPT, andAMS),theArthritisNationalResearchFoundationEngTanScholarAward(TBN),theArthritisFoundation(AMSandPMG),theKirklandScholarAward(LAC) andMeritAwardsfromtheUSDepartmentofVeteransAffairs(JBHandGSG).KoreaHealthcareTechnologyR&DProject(A010252,A080588;SCB)andWake ForestUniversityHealthSciencesCenterforPublicHealthGenomics(CDL),EuropeanScienceFoundation07-RNP-083(MAR)andFedericoWilhelmAgricola Foundation(BAPE).Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript. CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist. *E-mail:[email protected] Introduction populations are disproportionately affected by SLE [21] and healthdisparitiesinthesegroupsshowonsetatayoungerage[22]. TumourNecrosisFactorSuperfamily(TNFS)memberscontrol Hispanic and African-American populations have genomes wide-ranging facets of immunity when they interact with their which reflect recent admixture on ancient substructures [16]. complimentary TNF Receptors [1]. One of these, TNFSF4 Hispanic cohorts have rich diversity of source ancestry with (OX40L), uniquely binds its receptor, monomeric TNFRSF4 Southern European, Amerindian and West African contribution (OX40),onTlymphocytestostronglyactivateNF-kB[2].Several totheinheritedgenomeandtheforceddiasporaofAfricanstothe lines of evidence published over the last 15 years suggest the Americas also resulted in gene flow and two-way admixture TNFSF4–TNFRSF4 interaction is required for the induction of between previously reproductively isolated West African and anti-tumour immunity, allergy and autoimmunity [3–6] but also European ancestral populations [23]. African populations today inhibits generation of adaptive T regulatory (TR1) cells [7]. The tendtohaveshorterhaplotypesbecausetheyusuallyhaveancestors outcome is not limited to human disease; blockade of the who have experienced more recombination events without popu- TNFSF4-TNFRS4 interaction has ameliorative effects in animal lationbottlenecksorfoundereffectsinemigrantpopulations[24]. models of T cell pathologies [8] including allergic and autoim- Common shorter haplotypes are often subdivisions of the larger mune manifestations [9]. Genetic variation at TNFSF4 has been haplotypesfoundinnon-Africansandsocanbecorrelatedtothese associatedwiththeautoimmunediseasesystemiclupuserythema- [25].Inadmixedpopulations,thegeneticcomponentattributableto tosus (SLE),andother inflammatoryconditions includingathero- the West African ancestral population would equate to a faster sclerosis andischaemic stroke. decayofLD.ThebreakdownofLDisthereforegreaterinAfrican- SLE is the prototypic multi-system autoimmune disorder. Americans, because the majorcomponent(80% ormore) oftheir High-affinity, pathogenic IgG autoantibodies to an array of genome is West-African, compared to Hispanics who have nuclearantigensareahallmarkofpathogenesisandcharacterise componentestimatesbetween4–11%[23,26]. theglobal perturbation of theimmunesystem in SLE.Variation We infer a fine-scale map of the recombination rate and in at least 25 genetic loci with modest effect sizes are thought to location of hotspots within each entire population and in explain the genetic component of SLE [10]. The strong genetic subgroups of interest. We have used principal components (PC)- basis to disease is well-established and has been strengthened by based strategies to adjust for major ancestry before performing a the advent of GWAS, which has corroborated the association of high-resolution association study which utilises typed and proba- immunologically relevant loci with SLE [11–13]. We have bilisticgenotypestomaptheTNFSF4locus.Bysurveyingcommon previously shown single nucleotide polymorphisms (SNPs) in the variants up to 1000 Genomes Phase 1(v3), we aim to identify 59 TNFSF4 region to be associated with lupus in European commoncausalvariationatTNFSF4associatedwithSLE.Cross- families and a case-control cohort [4]. The increased association comparison of associated risk haplotypes across four populations of 59 risk alleles with disease has been replicated in East Asian focuses these analyses.The AAassociation replicates ina smaller populations [14,15], highlighting the genetic similarities at this cohort of AA-Gullah [27]. Our haplotype analyses find informa- locusintheseancestrallydistinctpopulations.MultipleSLErisk- tiverecombinantsinAfrican-AmericansandEuropeanstoresolve associated TNFSF4 variants are also associated with systemic genetic variantsassociated with SLEat thislocus. sclerosis [16], primary Sjo¨gren’s syndrome [17] and myocardial These data are used to perform six case-control association infarction [18,19]. studies and a trans-ancestral mapping experiment using in excess A major obstacle in the identification of disease-specific causal of 17900 subjects. We attempt to define causal variation at variantsatTNFSF4intheEuropeanandEastAsianSLEcohorts TNFSF4 in SLE susceptibility. In a complementary strategy we has been the strong linkage disequilibrium (R2.0.8) exhibited by perform association analysis using TNFSF4 alleles and lupus genotypedTNFSF4alleles,whichhasresultedinahighfrequency phenotypes. We explore the mechanism by which TNFSF4 extended haplotype associated with risk of disease instead of influences perturbation of the immune process in inflammatory delineating causal variations at the locus [4]. It is probable that disease. Finally, we interrogate risk alleles in terms of their migration out of Africa involved many founder effects and influence on transcription factor binding using a bioinformatics bottlenecks to increase haplotype length in East Asian and approach.Theresearchpresentedusestrans-ancestralmappingto European populations [20]. Hispanic and African-American inform thiscomplextrait. PLOSGenetics | www.plosgenetics.org 2 July2013 | Volume 9 | Issue 7 | e1003554 TransAncestralFineMappingofSLELocusTNFSF4 andcommonvariants (SNPsandINDELs)across thelocususing Author Summary MAF.1%intheimputationscaffold.Asdescribedinmethods,we Systemic lupus erythematosus (SLE/lupus) is a complex used a post imputation filter of HWE.0.01 and info .0.7 to disease in which the body’s immune cells cause inflam- include only genotyped and high quality imputed SNPs. The mation in one or more systems to cause the associated estimated concordance between imputed and true genotypes for morbidity. Hormones, the environment and genes are all theSNPs presented inthisstudy is0.95forall cohorts.Thefinal causalcontributorstoSLEandoverthepastseveralyears characteristics of alldatasets are presented in Table1. thegeneticcomponentofSLEhasbeenfirmlyestablished. Severalgeneswhichareregulatorsoftheimmunesystem Bayesian inference of fine-scale map of recombination are associated with disease risk. We have established one of these, the tumour-necrosis family superfamily member rates and hotspot densities at TNFSF4 4(TNFSF4)gene,asalupussusceptibilitygeneinNorthern The European sex-averaged and female-only recombination Europeans. A major obstacle in pinpointing the marker(s) maps generated by deCODE (http://www.decode.com/ atTNFSF4whichbestexplaintheriskofSLEhasbeenthe addendum/), are based on 15,257 and 8,850 directly observed strong correlation (linkage disequilibrium, LD) between recombinations,respectively.Thesemapshavearesolutioneffective adjacent markers across the TNFSF4 region in this down to 10kb and comparing them to the HapMap 3 and 1000 population.Toaddressthis,wehavetypedpolymorphisms Genomespopulation-averagedmaps[30,31],wefounddifferences inseveralpopulationsinadditiontotheEuropeangroups. attheTNFSF4locus.Thus,weestimatedbackgroundrecombina- The mixedancestry ofthesepopulations givesadifferent tion rates in AA, East Asians, Europeans and Hispanics using a LD pattern than that found in Europeans, presenting a Bayesiancomposite-likelihoodmethod.Theinclusionofahotspot method of pinpointing the section of the TNFSF4 region model allowed sampling of hotspots from the Markov chain and which results in SLE susceptibility. The Non-European inference of mean posterior hotspot densities from a threshold populations have allowed identification of a polymor- upwards of 0.25, giving a detection power of 50% and a false- phism likely to regulate expression of TNFSF4 to increase susceptibility to SLE. discoveryrateof4%[32].InAsians,EuropeansandHispanicsthe bulk of the recombination occurs in less than 5% of sequence (Figure1andFigure2).Anexceptiontothispatternisfoundin the African-American cohort, with increased recombination rate Results and higher density and proportion of hotspots across the locus To delineate the causal variation at TNFSF4, we genotyped (Figure1,Figure2).Inallpopulations,peakrecombinationisat SNPs in a 200Kb section of chromosome 1q25.1 encompassing the 59 boundary of the TNFSF4 gene and approximately 120kb TNFSF4 (23.6kb) and the 59 region (150 kb). Population into the 59 region. A difference in African-Americans is that stratification bias and effects due to admixture were addressed recombinationextends30kbfromtheTNFSF4geneboundaryinto using the approach of Namjou and colleagues [28]. We also the59region,whilstthereisnegligiblerecombinationinthissection genotyped 347 SNPs used by Halder [29] to correct for major in the other populations (Figure 1) and this is compatible with ancestry in each population as identified by a PCA-based increasedcomplexityofthegenomicregioninAfrican-Americans. approach (Supplementary Figure S1). As outlined in methods, SNPsandindividualsthatfailedqualitycontrolwerefiltered;pre- Single marker association of 59 TNFSF4 SNPs with SLE and post- imputation SNP counts and a description of the Theassociationdatapresentedareformarkersafterimputation component sample setsispresented inTable1. usingthe1000GenomesphaseIintegratedvariantsetv3(March To directly compare genotyped SNPs we used the phased 2012,NCBIbuild37)[30].TheTNFSF4locusiswellestablishedin chromosomes of1000Genomesphase I integrated variant setv3 SLE therefore we have presented uncorrected nominal p-values (March 2012, NCBI build37) (The 1000 Genomes Project forvariants.InEastAsians,EuropeansandHispanicsmanystrong Consortium) [30] and IMPUTE v2.2, together with second associations (P 1028,10216) at TNFSF4 are detected. Multiple u reference sets defined in Table 1. We imputed missing data susceptibility alleles inthe TNFSF4 59regionare overrepresented Table1. Populationdemographics and imputationreference data forSLE-control cohorts post QCfiltering. European EastAsian Hispanic AA-Gullah Cases Controls TOTAL Cases Controls TOTAL Cases Controls TOTAL Cases Controls TOTAL Males 344 1151 1495 167 225 392 119 73 192 136 593 729 Females 3088 2489 5577 1333 1171 2507 1229 644 1872 1541 1341 2882 Unknown 3 236 239 TOTAL 3432 3640 7072 1500 1396 2896 1348 717 2065 1680 2170 3850 SNPS(TYPED) 89 89 89 65 65 65 51 51 51 88 88 88 SNPS(ALL) 244 244 244 450 450 450 460 460 460 393 393 393 Imputationreference1 1000G 1000G 1000G 1000G 1000G 1000G 1000G 1000G 1000G 1000G 1000G 1000G Imputationreference2 OMNI-QUAD UK-CanadianGWAS Numbersafterfilteringforduplicates,FDRs,HWE,missingnessandmajorancestry.postSNPswithINFOscores,0.7excluded,SNPSwithHWE,0.01excluded. doi:10.1371/journal.pgen.1003554.t001 PLOSGenetics | www.plosgenetics.org 3 July2013 | Volume 9 | Issue 7 | e1003554 TransAncestralFineMappingofSLELocusTNFSF4 Figure1.FinescalemapsofrecombinationrateinferredfromEastAsian,European,HispanicandAfrican-Americancontrolphased chromosomes.1568randomlyassignedchromosomesfromeachgroupweretestedusingRhomap,fromtheLDHAT2.0package.Thefine-scale mapofrecombinationrate(4Ner/kb)wasinferredacross200kbofchromosome1q25encompassingTNFSF4geneandextended59and39regions. doi:10.1371/journal.pgen.1003554.g001 inSLEcases(Table2,Figure2).Intermsofsinglemarkers,best associated in African-Americans and the 59 association replicates evidenceofassociationwithdiseaseinEuropeansisobservedwith in a small cohort of AA-Gullah (Supplementary Table S1), rs2205960-T, 10kb 59 from the TNFSF4 gene (P=5.61610215, underpinning thisgeneas a global SLEsusceptibility gene. OR=1.34(95%CI1.25–1.44)).TheTalleleofrs2205960alsohas In African-Americans, the best evidence for the 59 SNP strongest association with Hispanic SLE (P=1.7610210, association with disease are from rs1234317-T (P=2.2861025, OR=1.65 (95% 1.42–1.91)). In Europeans, an additional 15 OR=1.4 (95%CI 1.25–1.56)) and rs2205960-T (P=7.261025, SNPs reach genome-wide significance (P,561028) most of these OR=1.48 (95%CI 1.22–1.67)) and rs1234314 –G riskallelesalsoreachthislevelofsignificanceintheEastAsianand (P=3.1161025,OR=1.22(95%CI1.13–1.32)).Thereisatrend Hispaniccohorts(Table2).Several59riskallelesassociatedwith for under-representation of the minor alleles of rs1234314-C, disease in East Asians, Europeans and Hispanics are also rs1234315-C,rs844642-G,rs844644-A,rs2795288-Tandrs844654- PLOSGenetics | www.plosgenetics.org 4 July2013 | Volume 9 | Issue 7 | e1003554 TransAncestralFineMappingofSLELocusTNFSF4 Figure 2. Single marker association atTNFSF4locus inA. East Asian,B. European,C. Hispanic,D. African-American SLE-control populations.Thestrengthoftheassociation(-log10uncorrectedP)ofmarkersacross240kbofchromosome1q25.ThisregionalplotdepictsTNFSF4 associationwithSLEversuschromosomalposition(kb)inEastAsians,Europeans,HispanicsandAfrican-Americanpopulations.Themostassociated variantsineachgrouparelabelled,asisthebest-associatedmeta-analysisSNPrs2205960.Markersarecolour-codedfortheircorrelationcoefficient (r2)valuesaccordingtothelegend. doi:10.1371/journal.pgen.1003554.g002 A in SLE cases resulting in a flipped OR for these variants sameimputationanalysisandsubjecttothesameQCastheSNPs (Table2). and probabilistic genotypes incorporated into our association analyses. We identify a deletion at rs200818062 [-/G] to be AssociationofintragenicTNFSF4singlemarkerswithSLE associated with SLE in all groups tested. This indel is located Examining the genetic association between SNPs within the 22.4 kbfromthestartsiteofthecommontranscript(Transcript1) TNFSF4 gene and SLE we identify association of rs1234313-G, of TNFSF4 and is in strong LD with (R2.0.8) rs1234317 and within intron1, with SLE in Asians (P=4.3761028, OR=1.38 rs2205960. (95%CI 1.32–1.44)), and Europeans (P=1.1161025, OR=1.15(95% CI 1.11–1.27). In both cohorts rs1234313-G is Best evidence meta-analysis partitioned from other associated SNPs by a recombination We used a logistic regression model fitted with an interaction hotspot at the TNFSF4- 59 boundary. However, correlation term (effect) in the R statistical package to investigate cross-study coefficient R2 values between this marker and risk-associated 59 heterogeneity. P-values for individual associated SNPs were variants suggest strong correlation. We identify under represen- generated using a likelihood-ratio test. We found no evidence of tationofrs10798265-AinAfrican-AmericanSLE(P=9.2461025, heterogeneityforthekeyrisk-haplotype-taggingcommonvariants 0.84(95%CI 0.78–0.9)). There is suggestion of additional modest which span the locus. Our null hypothesis - that all studies were association signals (P,1024) from a series of SNPs located at the evaluating the same effect size- held true for key variants TNFSF4-39UTRboundary inthesame cohort. associated with riskofSLE. We combined the association data for variants across the 59 Imputation of typed bi-allelic indels TNFSF4regioninEastAsians,Europeans,HispanicsandAfrican- Imputation gave 257 common (.1% MAF) bi-allelic indels at Americans, to more powerfully estimate the true effect size theTNFSF4locus,mostlyneutral.Theindelswereincludedinthe (Table 3). The average effect size across all datasets was PLOSGenetics | www.plosgenetics.org 5 July2013 | Volume 9 | Issue 7 | e1003554 TransAncestralFineMappingofSLELocusTNFSF4 5–0.81) 8–0.84) –1.63) 2–1.69) 51–1.88) 62–0.90) 61–0.88) 2–0.89) 9–1.66) 5–0.80) 42–1.70) 5–1.61) 8–1.65) 1–1.57) 8–1.54) 3–1.04) 54–0.8) 1–0.74) 7–1.64) 37–1.64) 37–1.64) 2–1.58) 38–1.64) 7–1.56) 37–1.64) 38–1.64) 8–1.64) 8–1.66) 34–1.60) 34–1.60) 37–1.63) 33–1.59) 33–1.59) 48–1.61) Hisp 0.68(0.5 0.71(0.5 1.5(1.36 1.56(1.4 1.69(1. 0.76(0. 0.74(0. 0.75(0.6 1.53(1.3 0.67(0.5 1.56(1. 1.48(1.3 1.51(1.3 1.44(1.3 1.41(1.2 0.79(0.5 0.67(0. 0.67(0.6 1.51(1.3 1.50(1. 1.50(1. 1.45(1.3 1.51(1. 1.41(1.2 1.51(1. 1.51(1. 1.51(1.3 1.52(1.3 1.47(1. 1.47(1. 1.50(1. 1.46(1. 1.46(1. 1.48(1.3 4) 0) 1) 0) 5) 5) 5) 5) 5) 5) 6) 26) 5) 6) 6) 87) 88) 34) 40) 1.3 0.9 0.9 91) 36) 85) 1.3 26) 30) 35) 24) 92) 0.8 83) 35) 1.3 1.3 35) 1.3 28) 1.3 1.3 6) 33) 1.2 –1. 1.3 1.2 1.2 36) Eur 0.83(0.78–0. 0.82(0.76–0. 1.27(1.20–1. 1.33(1.26–1. 1.26(1.18– 0.84(0.77– 0.84(0.78– 0.84(0.78–0. 1.29(1.22–1. 0.81(0.78–0. 1.23(1.17– 1.19(1.13–1. 1.23(1.17–1. 1.28(1.22–1. 1.18(1.11–1. 0.85(0.79–0. 0.81(0.78– 0.78(0.74–0. 1.28(1.21–1. 1.28(1.21– 1.28(1.21– 1.29(1.22–1. 1.28(1.21– 1.21(1.14–1. 1.28(1.21– 1.28(1.21– 1.2(1.13–1.2 1.26(1.19–1. 1.19(1.13– 1.197(1.14 1.28(1.21– 1.19(1.13– 1.19(1.13– 1.28(1.21–1. CI) As 0.72(0.67–0.87) 0.72(0.68–0.78) 1.37(1.26–1.48) 1.43(1.32–1.54) 1.55(1.40–1.69) 0.71(0.66–0.78) 0.71(0.67–0.77) 0.71(0.67–0.77) 1.37(1.26–1.48) 0.73(0.68–0.79) 1.37(1.26–1.48) 1.39(1.29–1.50) 1.37(1.26–1.48) 1.33(1.23–1.43) 1.38(1.28–1.48) 0.73(0.68–0.81) 0.73(0.68–0.79) 0.74(0.68–0.81) 1.36(1.25–1.47) 1.36(1.25–1.47) 1.34(1.23–1.45) 1.34(1.24–1.44) 1.34(1.23–1.45) 1.40(1.29–1.52) 1.35(1.24–1.46) 1.37(1.26–1.48) 1.43(1.31–1.55) 1.34(1.23–1.45) 1.377(1.27–1.49) 1.38(1.27–1.48) 1.36(1.25–1.47) 1.37(1.26–1.48) 1.37(1.26–1.48) 1.36(1.25–1.47) ohorts. OddsRatio(95% AAG 0.82(0.76–0.88) 1.02(0.91–1.13) 1.4(1.25–1.56) 1.48(1.2–1.67) 1.45(1.28–1.63) 1.03(0.90–1.16) 1.07(0.93–1.22) 1.00(0.88–1.13) 1.44(1.23–1.65) 0.98(0.89–1.07) 1.38(1.22–1.54) 1.00(0.91–1.11) 1.29(1.15–1.43) 1.05(0.95–1.15) 1.02(0.92–1.12) 0.98(0.91–1.05) 1.02(0.93–1.12) 0.8(0.72–0.90) 1.26(1.12–1.39) 1.26(1.12–1.39) 1.25(1.12–1.39) 1.04(0.94–1.14) 1.26(1.12–1.39) 0.95(0.85–1.06) 1.26(1.12–1.39) 1.27(1.13–1.40) 1.17(1.05–1.29) 1.30(1.10–1.50) 1.18(1.06–1.3) 1.17(1.057–1.29) 1.27(1.14–1.41) 1.18(1.06–1.30) 1.18(1.06–1.30) 1.28(1.15–1.42) (Hisp)SLE-controlc EurHisp 2299662.43103.6710 22107662.91103.0610 22119661.77103.6510 221510665.61101.7010 2298662.54103.3810 2274661.09101.7410 2275661.12102.6210 2275661.04103.0310 221210662.48104.7110 22119669.2101.8510 221010661.7610310 2289669.40103.8210 221010661.79108.410 22138662.59103.0710 2267661.35102.0810 2662.08100.07 22119666.05102.3610 22129665.87106.9110 22129663.83101.2310 22129662.69101.4110 22129662.68101.4110 22138662.22102.9310 22129662.89101.1910 2276661.86102.9210 22129664.53101.2510 22129664.91101.210 22810669.21108.7710 22109664.36103.7510 2289666.04104.1910 2289666.08104.1710 22129664.78101.5110 2289667.82108.9410 2289667.8108.9210 22129662.87107.3710 anic e 211 210 28 210 290 2100 2110 211 28 210 280 29 28 28 210 29 280 28 280 270 290 28 280 270 29 290 280 280 280 2110 210 dHisp p-valu As 65.8410 61.7810 61.8510 61.1810 65.451 62.091 63.981 62.5610 61.3810 65.6210 61.271 61.3610 62.1110 65.1710 66.0310 61.4310 69.471 63.0810 63.301 61.461 64.581 68.2810 61.881 63.161 65.4310 65.471 64.791 61.301 61.301 65.841 61.7810 n d an(Eur)a Unadjuste AAG 2563.1110 0.7 2562.2810 2567.2010 2562.8510 0.65 0.35 0.91 2466.1310 0.6 2566.8210 0.89 2463.0510 0.34 0.68524 0.74281 0.7133 2361.2810 2468.9210 2468.1210 2469.5310 0.47 2469.6910 0.37 2469.0110 2465.9310 0.01 236910 2367.2910 2368.7710 2465.8210 2366.5410 2366.5510 2463.9110 e p o B s),Eur AA/A C/G C/T T/C T/G 7D/I G/T 8G/A A/C A/G T/A 8T/C T/G G/T G/A C/A A/G 9A/T C/A C/T 9T/C 9A/G C/T 9T/C T/A 9T/A T/C T/C A/G 7C/T 7C/T T/C 7T/C 7T/G G/A A 8 4 9 7 1 4 3 9 8 2 7 9 5 4 8 9 2 2 4 2 6 2 3 6 9 3 3 3 3 3 Asian( Hisp 0.42/0.4 0.36/0.4 0.38/0.2 0.37/0.2 0.24/0. 0.34/0. 0.33/0. 0.33/0.4 0.38/0.2 0.42/0.4 0.38/0. 0.46/0.3 0.39/0.2 0.44/0.3 0.5/0.42 0.09/0.1 0.42/0. 0.29/0.3 0.39/0.2 0.39/0. 0.39/0. 0.43/0.3 0.39/0. 0.45/0.3 0.39/0. 0.39/0. 0.46/0.3 0.39/0.2 0.46/0. 0.46/0. 0.39/0. 0.46/0. 0.46/0. 0.39/0.3 3 7 3 7 3 3 3 4 4 4 4 4 4 4 SinglemarkerassociationresultsforEastTable2. F_AA/F_ABMarkerCoordinate AAGAsEur rs1234314173.17740.31/0.360.38/0.70.43/0.43 rs1234315173.17850.2/0.20.39/0.470.47/0.48 rs1234317173.18780.11/0.080.32/0.250.31/0.26 rs2205960173.19150.07/0.050.32/0.250.27/0.22 rs200818062173.19890.1/0.070.26/0.20.27/0.2 rs6673550173.19910.16/0.160.48/0.40.45/0.5 rs844642173.20780.11/0.120.41/0.490.43/0.4 rs844644173.20950.16/0.160.41/0.490.43/0.47 rs12039904173.21230.06/0.040.32/0.250.28/0.23 rs2795288173.21390.47/0.480.41/0.490.41/0.47 rs4916315173.21540.12/0.090.31/0.250.28/0.2 rs1012507173.21950.28/0.280.34/0.270.38/0.34 rs35086785173.22020.13/0.110.31/0.250.28/0.23 rs844648173.22430.29/0.280.42/0.360.33/0.28 rs844649173.22510.33/0.320.44/0.360.43/0.39 rs844651173.23150.15/0.150.49/0.420.43/0.47 rs1099447173.23280.47/0.470.41/0.490.42/0.4 rs844654173.23610.12/0.150.25/0.310.23/0.28 rs10489265173.23670.14/0.120.31/0.250.29/0.23 rs34313362173.24070.14/0.120.31/0.250.29/0.2 rs10912576173.24190.14/0.120.31/0.250.29/0.2 rs844663173.24360.28/0.270.42/0.350.33/0.28 rs12403570173.24440.14/0.120.31/0.250.28/0.2 rs12049190173.24730.29/0.30.33/0.260.38/0.33 rs35634597173.24820.14/0.120.31/0.250.29/0.2 rs67638449173.24930.14/0.120.31/0.250.29/0.2 rs12750070173.24980.19/0.170.33/0.250.39/0.34 rs12405577173.25000.07/0.050.32/0.260.29/0.24 rs6697570173.25410.19/0.160.33/0.260.38/0.3 rs12143114173.25450.2/0.170.33/0.260.38/0.3 rs35691278173.25510.14/0.110.31/0.250.29/0.2 rs11802979173.25630.19/0.160.32/0.250.38/0.3 rs10912579173.25630.19/0.160.32/0.250.38/0.3 rs10912580173.25660.14/0.110.31/0.250.29/0.24 Variantsinboldareimputedusing1000Genomesphase1(v3).doi:10.1371/journal.pgen.1003554.t002 PLOSGenetics | www.plosgenetics.org 6 July2013 | Volume 9 | Issue 7 | e1003554 TransAncestralFineMappingofSLELocusTNFSF4 or 4 1 3 4 3 2 1 rs 0, 14 596 3 0 4 2 3 2 120 rs rs96 23 23 23 on pValuers2205+ 1 64.5710 0.12 1 0.02 0.14 0.13 0.12 0.13 0.10 0.10 0.09 0.08 0.03 0.07 0.01 0.03 0.13 0.11 0.16 0.17 0.15 66.6110 0.10 0.10 69.8610 0.02 0.05 0.04 0.08 0.06 0.12 0.08 0.37 ditioned n o c n, o ati ci o oned SNPass conditi34314 214 28 210 29 29 29 29 29 29 29 29 29 28 29 28 27 26 26 24 24 25 25 24 24 25 23 24 23 24 23 23 23 nominal pValuers12on 64.1210 62.5710 62.3110 1 62.6410 61.2510 61.6710 61.9510 62.6310 62.6210 64.4810 64.8210 65.1410 61.1610 63.4910 61.4110 62.7410 66.6810 61.8310 61.0510 61.3410 65.2110 64.8210 61.8610 61.8610 67.7410 64.7810 66.0910 63.5310 62.2810 67.5110 67.9110 67.2910 0.06 eforthe u al v P d e ail o-t w oned andt pValueconditirs2205960on 1 0.04 0.12 2763.8110 0.01 0.10 0.08 0.07 0.09 0.06 0.06 0.05 0.05 0.02 0.04 2367.0910 0.03 0.02 0.01 0.24 0.25 0.16 2567.7410 0.14 0.13 2565.8010 2461.1410 0.17 2466.610 0.16 2361.7910 0.04 2363.3410 0.03 uencies(FREQ1) q e 4SNPs. pValue 23461.7110 22861.1610 22862.0610 22866.5210 22868.8510 22869.3910 22761.3610 22761.9710 22762.2010 22762.2210 22765.4610 22765.6510 22765.9410 22661.7110 22663.8910 22664.5610 22261.2410 22261.7010 22261.7410 22161.1310 22161.5210 22162.0010 22162.6010 22163.6910 22163.7010 22163.7410 21961.4310 21764.0310 21764.4710 21663.3310 21669.5910 21561.2710 21562.6510 21163.0210 udingallelefr F cl S n eforTNF WEIGHT 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 ysisresultsi u al Pval SE 0.014 0.066 0.097 0.108 0.125 0.137 0.067 0.070 0.070 0.074 0.070 0.038 0.096 0.067 0.067 0.067 0.081 0.042 0.043 0.068 0.046 0.090 0.067 0.082 0.082 0.063 0.084 0.044 0.118 0.078 0.044 0.154 0.082 0.010 eta-an m ciation FREQ 0.549 0.757 0.775 0.396 0.615 0.637 0.755 0.240 0.247 0.764 0.247 0.663 0.767 0.247 0.245 0.860 0.684 0.396 0.323 0.248 0.420 0.779 0.755 0.688 0.688 0.757 0.249 0.661 0.375 0.684 0.672 0.339 0.314 0.547 mnslist o u ass AB col the AA/ G/T C/T C/T C/G C/T C/T C/T T/C A/G C/T A/T G/A C/T T/G A/C I/D C/T T/C T/C T/C T/C C/T T/A G/T C/T T/A T/C G/T A/G T/A C/A A/C T/G G/A extsix of n e videncemeta-analysis COORDINATE(Mb) 173.1915 173.1878 173.2123 173.1774 173.2154 173.2493 173.2407 173.2367 173.2566 173.2551 173.2482 173.2419 173.2444 173.2202 173.2361 173.1988 173.2500 173.2243 173.2436 173.2541 173.2545 173.2498 173.2139 173.2563 173.2563 173.2328 173.1785 173.2195 173.2078 173.2473 173.2095 173.2251 173.1991 173.1915 nslistSNPcharacteristics,thrs1234314rs2205960.+gen.1003554.t003 Beste AME ecolumonbothournal.p Table3. MARKERN rs2205960 rs1234317 rs12039904 rs1234314 rs4916315 rs67638449 rs10912576 rs34313362 rs10912580 rs35691278 rs35634597 rs12046550 rs12403570 rs35086785 rs10489265 rs200818062 rs12405577 rs844649 rs844663 rs6697570 rs12143114 rs12750070 rs2795288 rs10912579 rs11802979 rs844654 rs1234315 rs1012507 rs844642 rs12049190 rs844644 rs844651 rs6673550 rs1099447 Thefirstthreconditioneddoi:10.1371/j PLOSGenetics | www.plosgenetics.org 7 July2013 | Volume 9 | Issue 7 | e1003554 TransAncestralFineMappingofSLELocusTNFSF4 Figure3.LDplotsatTNFSF4locusinfourpopulations.Thissectionofchromosome1q25.1encompassestheTNFSF4geneandupstream regionasdefinedbycustomalgorithminHaploview4.2.ThemeasureofLDwasusedtodepict57SNPscommontoallcohorts,postQCand1000 genomes imputation. The pair-wise correlations between TNFSF4 markers is illustrated in these plots by the correlation coefficient R2(where r2=0=no correlation, white; 0,R2,1, gradations of grey; R2=1=complete correlation, black). The TNFSF4 gene is positioned above the plots relativetohaplotypeblocks(blacktriangles)andgreyticksindicateSNPlocationstoscale. doi:10.1371/journal.pgen.1003554.g003 computedusinginversevarianceweightingofeachstudy.Wefind Conditional regression analysis of 59 single-markers the 59 association of TNFSF4 with SLE greatly reinforced. As expected, our 59 association data suggest pairwise LD rs2205960-T, the most associated allele in Europeans and betweenmarkersisweakestinAfricanAmericansandstrongestin Hispanics, (P=1.71610234, OR=1.43[1.26–1.60]), and Asians.Inordertoestablish whetherthesignalsidentifiedbyour rs1234317-T (P=1.16610228, OR=1.38[1.24–1.54]) have the trans-ancestralfine-mappingexperimentrepresentcausalvariants, strongest combined association with disease (Table 3). These independent risk factors, or if we have simply found surrogate adjacent markers are separated by 3 kb of chromosome 1. Allele markers strongly correlated with causal variants, we conditioned frequencies for rs2205960 for 1000 Genomes populations are theassociationdatafromeachpopulationwiththemarkerwhich presented insupplementary data. represented thebest evidenceofassociation. PLOSGenetics | www.plosgenetics.org 8 July2013 | Volume 9 | Issue 7 | e1003554 TransAncestralFineMappingofSLELocusTNFSF4 Inallpopulations,rs2205960-T,arisk-haplotypetagSNPwith 0 6 highp-valueandeffectsize(Table3,Figure3)isassociatedwith 9 05 SLE; a similar trend is illustrated by the adjacent marker rs22 0.009 0.94 21 trhs1e2s3i4gn31al7a-Ttr.s1C2o3n4d3i1ti7onisinlogstoanftersr1c2o3n4d3i1ti7onoinrgrsf2o2r0r5s29260059w6e0,fianndd 7 this is consistent for across populations (Table 4). If the reverse 1 Hispanic rs12343 0.005 21 0.010 arEsn1ua2rlo3yp4sie3sa1n7iss,atpnhederrfHeoriismspeardnesicidasnu(Padl=wa9se6so1cc0ioa2nti4doAinSti,oPant,r1os20n2204t5hE9Ue6,0Ppr=iens0e.An0c1sei5aHnois)f., n/ In all apart from the AA group, conditioning on rs1234317 or a 4 Amerindi p-value rs123431 21 0.007 2463.5610 rrss22220055996600,lres1a2ve3s43a17reasniddurasl12s3ig4n3a1l4aitnrtsh1e2s3e4a3n1a4l.ysWes.e included Conditional analysis of meta-analysis data 960 m. We find conditioning on the presence of rs1234317 or 05 dig rs2205960, association of intron 1 markers tested for all groups rs22 0.038 0.57 21 para (iTs alobslte, 2c)o.nWfiremailnsogctohnedseitioansedsetchoenmdaertya-atnoal5y9sisraissksocaisastoiocinatdioantas 34317 25610 equentist o(Pn=3rs.28210651906207),alnodcatfeoduantdthereTsiNduFaSlF4a-5ss9obcioautinodnarya.tTrhse12r3ev4e3r1s4e rs12 0.017 21 4.88 dafr (aPn=aly4s.i1s26fo1u0n2d14).inTcrheeasseeddatraesisduugaglestastswocoiaitniodnepeantderns2t2s0ig5n9a6l0s n European p-value rs1234314 21 2568.9810 2864.020 onalmodela arwsn1itd2h3r4sin232c1r04e5ai9ns6eSd0LraEesms.oCocvoiaentdidoiantisosanoncindiagteitofhfneecamtteaTttaN-rdsF2aS2tFa045o9(nT6b0aobctlhoemrs3p1)a2.r3e4d31to4 5960 gadditi Mcoohdoerltlsing the pattern of inheritance in independent rs220 0.013 0.024 21 edusin assIonciagteinonotyopfe-bTaNseFdSFa4nawlyistehs,StLhEe maroedetlshethaadtdbiteisvte/fidtsomthinea5n9t 17 elect models. 3 s rs1234 246410 21 246910 Pvalues Hhaapplloottyyppeesbifurcation of TNFSF4 risk and non-risk groups. Asian p-value rs1234314 21 0.190 0.008 gframework. ioddfieaHngLrtaiaDpfmileodstoyffpoferraossemsasoicgchinapiptfheiocadpaseundhltalaytpioglaonesnt.syooTpcteyoiasp,tbeesedwtteewfroirtcvhoisnuersaaitsclrkiuhsecottefchdoedhibsboerirafetusaerkctdaewostitewoernnde TNFSF4variantsinfour rs1234317rs2205960 0.0880.103 210.084 20.2241 uscovariatewithinaclusterin tTmtdrT(Fhhsi1Nhseeeie2getFaqf3luiSi-lug4noraFui3enecl4ri1aabeig4tn4lriye,(io,lusneanitmreasbh,-iceoe5sahl(f9lLkreemrpbD)ds.lo1a,oo)Tu2lcstast3niohtrc4decoipm3naurrp1rclraoay7relrxoreskaitpaaecmsnsonoddianindrloleldwgunrsfhsgsdtrt2eotioircos2namtht0otahte5nthiewsy9cetpduh6eheniss0ieacedu,ighdnbmrbSraahbebeNsmasoaetpttP-kr.hhaldoTeosodtlsfhcoyowiopercscnearaeitceamthetbtoviiedprocfadaklnrelnniaissinacnetfnkhwsrsatotteihhgstmionh)eeef. 9Conditionalregressionresultsfor5Table4. AAGullah+MarkerA1A2Coordinate p-value rs1234314 2rs1234314GC1731773921 rs1234317TC1731877750.004 rs2205960TG1731914750.001 ConditionalanalysesinSNPTESTv2CaseControl.Continuodoi:10.1371/journal.pgen.1003554.t004 thrAdoCTt(ats(FThhiunifoaoNofseeiprapTdSwntgnFkiphlbichhnucogSso)neal,oaetnrieFreonoynprTfteir4nf-npoefilv5nAiNo-Lleoc4lar)gtlti,maFon.Doh,iyntsnwS-LtpeticknerohhtFoeetoruiloeeshyn4ihn,dwkrcssneaieabaltlsbo-pihoihsrnrknrnAbsyelcaafesoioeciu.npAsfsTtHcraoiusclykniiWoNmnsahrapiedg)ktsct.crFeeynbrpieeaevsheCSopcdiathinasuiFoednifomoonhaptipm4uinnnrrmtcdalo,aaeiotdpspoihrstdlotclitaasalnlynaaaaozybssipndrtpytnsttareao(iyeidgidasSnnnlc.pboontbiguagdieEouvtttcrtepsssytieurhesbsnhtsiappatrr1meghersokfilje2ineieucepdinrc3llonmeeoaofkds4oEpicacrwnntns3uotnearogeclni1unmiosnoss-inp7t,dsrdccntocbauaeataAswrafiinnutlnnnerniasigLtdoardtayidteehaesnweDtiaenioplroiecddh.oasFstinnah2chioftFisst(rs2mgtFhtprioteta0nhauoocirhorn5gtgeotrpoezec9tsumebushype6bieruglneo0bracfeosterS.ttitrrssnoiiaii3ooi4mssaikatnnn)kky,--t, PLOSGenetics | www.plosgenetics.org 9 July2013 | Volume 9 | Issue 7 | e1003554 TransAncestralFineMappingofSLELocusTNFSF4 Figure 4. Haplotype Bifurcation Diagrams ofTNFSF4 andTNFSF4 for Four Populations. Plots are constructed using phased risk non-risk haplotypesfora.EastAsians,b.Europeans,c.Hispanicsandd.African-AmericansandillustratebreakdownofLDwithincreasingdistancesfromacore proximalTNFSF4SNPandareapproximatelytoscale.ThecoreislocatedattheTNFSF4gene-59boundary(blackcircle)andisselectedasthemost proximal59marker,rs1234314,ineachpopulation.Genelocationisdepictedtoscale;wehaveadditionallylabelledeachplottoshowthelocationof rs1234317andrs2205960,thebest-associatedmarkersfromthemeta-analysis. doi:10.1371/journal.pgen.1003554.g004 the TNFSF4-59 boundary in East Asians, Europeans, and population,whilstTNFSF4 isthemostfrequenthaplotypefor OR,1 Hispanics allow for the construction of near-identical haplotype all cohorts testedbut underrepresented in SLEindividuals. blocks including risk and non-risk haplotypes (designated The risk haplotype found in East Asians, Europeans and TNFSF4 and TNFSF4 , respectively)(Figure 3) which HispanicsisfragmentedintheAfrican-Americancohort;themost OR.1 OR,1 extend approx. 80kb into the TNFSF4 59 region. Multiple associated risk haplotype is 11kb (P=2.1261025, OR=1.52). associated risk alleles uniquely tag TNFSF4 , the risk Thishaplotypeblockextendsfromrs1234317tothebi-allelicindel OR.1 haplotype, which is overrepresented in SLE individuals in each rs200818062. Only one allele uniquely tags this haplotype, PLOSGenetics | www.plosgenetics.org 10 July2013 | Volume 9 | Issue 7 | e1003554

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Jennifer A. Kelly12, Javier Martin18, Joan T. Merrill19, Kathy L. Moser12, Timothy B. Niewold20, Lopez-Neyra, Consejo Superior de Investigaciones Cientificas, Granada, . Trans Ancestral Fine Mapping of SLE Locus TNFSF4.
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Trans-Ancestral Studies Fine Map the SLE-Susceptibility Locus TNFSF4 PDF

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Trans-Ancestral Studies Fine Map the SLE-Susceptibility Locus TNFSF4 Harinder Manku1, Carl D. Langefeld2, Sandra G. Guerra3, Talat H. Malik4, Marta Alarcon-Riquelme5, Juan-Manuel Anaya6, Sang-Cheol Bae7, Susan A. Boackle8, Elizabeth E. Brown9, Lindsey A. Criswell10, Barry I. Freedman11, Patrick M. Gaffney12, Peter A. Gregersen13, Joel M. Guthridge12, Sang-Hoon Han7, John B. Harley14, Chaim O. Jacob15, Judith A. James12,16, Diane L. Kamen17, Kenneth M. Kaufman14, Jennifer A. Kelly12, Javier Martin18, Joan T. Merrill19, Kathy L. Moser12, Timothy B. Niewold20, So-Yeon Park7, Bernardo A. Pons-Estel21, Amr H. Sawalha22, R. Hal Scofield12,16, Nan Shen23, Anne M. Stevens24, Celi Sun12, Gary S. Gilkeson25, Jeff C. Edberg25, Robert P. Kimberly26, Swapan K. Nath12, Betty P. Tsao27, Tim J. Vyse1* 1Department of Medical & Molecular Genetics, King’s College London School of Medicine, Guy’s Hospital, London, United Kingdom, 2Wake Forest School of Medicine,Winston-Salem,NorthCarolina,UnitedStatesofAmerica,3CentreforRheumatology&ConnectiveTissueDiseases,RoyalFree&UniversityCollegeMedical School,London,UnitedKingdom,4DivisionofImmunologyandInflammation,ImperialCollege,London,UnitedKingdom,5CentroPfizer-UniversidaddeGranada- Junta de Andaluc´ıa de Geno´mica e Investigaciones Oncolo´gicas, Granada, Spain, 6Center for Autoimmune Diseases Research, Universidad del Rosario, Bogota, Colombia,7HospitalforRheumaticDiseases,HanyangUniversity,Seoul,SouthKorea,8DivisionofRheumatology,UniversityofColoradoDenver,Aurora,Colorado, UnitedStatesofAmerica,9DepartmentofEpidemiology,UniversityofAlabamaatBirmingham,Birmingham,Alabama,UnitedStatesofAmerica,10RosalindRussell MedicalResearchCenterforArthritis,UniversityofCaliforniaSanFrancisco,SanFrancisco,California,UnitedStatesofAmerica,11DepartmentofInternalMedicine, WakeForestSchoolofMedicine,Winston-Salem,NorthCarolina,UnitedStatesofAmerica,12ArthritisandClinicalImmunologyResearchProgram,OklahomaMedical ResearchFoundation,OklahomaCity,Oklahoma,UnitedStatesofAmerica,13TheRobertS.BoasCenterforGenomicsandHumanGenetics,FeinsteinInstitutefor MedicalResearch,NorthShoreLIJHealthSystem,Manhasset,NewYork,UnitedStatesofAmerica,14DivisionofRheumatology,CincinnatiChildren’sHospitalMedical Centre,Cincinnati,Ohio,UnitedStatesofAmerica,15TheLupusGeneticsGroup,DepartmentofMedicine,UniversityofSouthernCalifornia,LosAngeles,California, United States of America, 16Department of Medicine, University of Oklahoma Healthy Sciences Center, Oklahoma City, Oklahoma, United States of America, 17DivisionofRheumatology,MedicalUniversityofSouthCarolina,Charleston,SouthCarolina,UnitedStatesofAmerica,18InstitutodeParasitologiayBiomedicina Lopez-Neyra,ConsejoSuperiordeInvestigacionesCientificas,Granada,Spain,19ClinicalPharmacology,OklahomaMedicalResearchFoundation,OklahomaCity, Oklahoma,UnitedStatesofAmerica,20DivisionsofRheumatologyandImmunology,MayoClinic,Rochester,Minnesota,UnitedStatesofAmerica,21Sanatorio Parque,Rosario,Argentina,22DivisionofRheumatology,DepartmentofInternalMedicine,UniversityofMichigan,AnnArbor,Michigan,UnitedStatesofAmerica, 23Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai, China, 24Center for Immunity and Immunotherapies, Seattle Children’s ResearchInstitute,Seattle,Washington,UnitedStatesofAmerica,25DivisionofRheumatologyandImmunology,MedicalUniversityofSouthCarolina,Charleston, SouthCarolina,UnitedStatesofAmerica,26DivisionofClinicalImmunologyandRheumatology,DepartmentofMedicine,UniversityofAlabamaatBirmingham, Birmingham,Alabama,UnitedStatesofAmerica,27DivisionofRheumatology,DepartmentofMedicine,DavidGeffenSchoolofMedicineatUCLA,LosAngeles, California,UnitedStatesofAmerica Abstract Wepreviouslyestablishedan80 kbhaplotypeupstreamofTNFSF4asasusceptibilitylocusintheautoimmunediseaseSLE. SLE-associated alleles at this locus are associated with inflammatory disorders, including atherosclerosis and ischaemic stroke.InEuropeans,the TNFSF4causalvariantshaveremained elusiveduetostronglinkagedisequilibriumexhibitedby allelesspanningtheregion.Usingatrans-ancestralapproachtofine-mapthelocus,utilising17,900SLEandcontrolsubjects includingAmerindian/Hispanics(1348cases,717controls),African-Americans(AA)(1529,2048)andbetterpoweredcohorts of Europeans and East Asians, we find strong association of risk alleles in all ethnicities; the AA association replicates in African-American Gullah (152,122). The best evidence of association comes from two adjacent markers: rs2205960-T (P=1.71610234, OR=1.43[1.26–1.60]) and rs1234317-T (P=1.16610228, OR=1.38[1.24–1.54]). Inference of fine-scale recombinationratesforallpopulationstestedfindsthe80kbriskandnon-riskhaplotypesinallexceptAfrican-Americans. Inthis populationthedecayofrecombination equatestoan11kbrisk haplotype,anchoredinthe 59regionproximal to TNFSF4andtaggedbyrs2205960-Tafter1000Genomesphase1(v3)imputation.Conditionalregressionanalysesdelineate the59risksignaltors2205960-Tandtheindependentnon-risksignaltors1234314-C.Ourcase-onlyandSLE-controlcohorts demonstraterobustassociationofrs2205960-Twithautoantibodyproduction.Thers2205960-Tispredictedtoformpartofa decamericmotifwhichbindsNF-kBp65withincreasedaffinitycomparedtors2205960-G.ChIP-seqdataalsoindicateNF-kB interactionwiththeDNAsequenceatthispositioninLCLcells.Ourresearchsuggestsassociationofrs2205960-TwithSLE across multiple groups and an independent non-risk signal at rs1234314-C. rs2205960-T is associated with autoantibody productionandlymphopenia.OurdataconfirmaglobalsignalatTNFSF4andarolefortheexpressedproductatmultiple stages of lymphocyte dysregulation during SLE pathogenesis. We confirm the validity of trans-ancestral mapping in a complex trait. PLOSGenetics | www.plosgenetics.org 1 July2013 | Volume 9 | Issue 7 | e1003554 TransAncestralFineMappingofSLELocusTNFSF4 Citation: Manku H, Langefeld CD, Guerra SG, Malik TH, Alarcon-Riquelme M, et al. (2013) Trans-Ancestral Studies Fine Map the SLE-Susceptibility Locus TNFSF4.PLoSGenet9(7):e1003554.doi:10.1371/journal.pgen.1003554 Editor:MarkI.McCarthy,UniversityofOxford,UnitedKingdom ReceivedOctober22,2012;AcceptedApril23,2013;PublishedJuly18,2013 Copyright: (cid:2)2013 Manku et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding:ThisworkwasmadepossiblethroughtheAllianceforLupusResearch(ALR)GenesfromSLEGEN:TheLupusGeneticsConsortium.Additionalsupport forthisworkwasobtainedfromWellcomeTrustprogrammegrant085492(TJV,HM),WellcomeTrustprojectgrant083167(TJV,HM),PO1AR49084(RPKandEEB), R01AR33062(RPK),R01AR62277(JBHandKLM),R01AR043274(KLM),R01AI063274(PMG),R37AI24717(JBH),R01AR042460(JBH),P01AI083194(JBH),P20 RR020143(JBH),P30AR055385(EEB),K08AI083790(TBN),LRPAI071651(TBN),R01CA141700(MAR),RC1AR058621(MAR),UL1RR024999(TBN),R01AR051545 (AMS),P30AR053483(JAJandJMG),U19AI082714(JAJandJMG),P30GM103510(JAJandJMG)andU01AI101934(JAJ)R21AI070304(SAB),R01AR43814(BPT), P60AR053308(LAC),M01RR-00079(LAC),R01AR057172(COJ),P60-AR049459(GSGandDLK),UL1-RR029882(GSGandDLK)andCTSAGrantNumberIULI RR025014-02(AMS),AI094377(SKN),AR060366(SKN).ThisstudywasalsosupportedbytheArthritisResearchUK(SGG,TJV),LupusResearchInstitute(TBN,BPT, andAMS),theArthritisNationalResearchFoundationEngTanScholarAward(TBN),theArthritisFoundation(AMSandPMG),theKirklandScholarAward(LAC) andMeritAwardsfromtheUSDepartmentofVeteransAffairs(JBHandGSG).KoreaHealthcareTechnologyR&DProject(A010252,A080588;SCB)andWake ForestUniversityHealthSciencesCenterforPublicHealthGenomics(CDL),EuropeanScienceFoundation07-RNP-083(MAR)andFedericoWilhelmAgricola Foundation(BAPE).Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript. CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist. *E-mail:[email protected] Introduction populations are disproportionately affected by SLE [21] and healthdisparitiesinthesegroupsshowonsetatayoungerage[22]. TumourNecrosisFactorSuperfamily(TNFS)memberscontrol Hispanic and African-American populations have genomes wide-ranging facets of immunity when they interact with their which reflect recent admixture on ancient substructures [16]. complimentary TNF Receptors [1]. One of these, TNFSF4 Hispanic cohorts have rich diversity of source ancestry with (OX40L), uniquely binds its receptor, monomeric TNFRSF4 Southern European, Amerindian and West African contribution (OX40),onTlymphocytestostronglyactivateNF-kB[2].Several totheinheritedgenomeandtheforceddiasporaofAfricanstothe lines of evidence published over the last 15 years suggest the Americas also resulted in gene flow and two-way admixture TNFSF4–TNFRSF4 interaction is required for the induction of between previously reproductively isolated West African and anti-tumour immunity, allergy and autoimmunity [3–6] but also European ancestral populations [23]. African populations today inhibits generation of adaptive T regulatory (TR1) cells [7]. The tendtohaveshorterhaplotypesbecausetheyusuallyhaveancestors outcome is not limited to human disease; blockade of the who have experienced more recombination events without popu- TNFSF4-TNFRS4 interaction has ameliorative effects in animal lationbottlenecksorfoundereffectsinemigrantpopulations[24]. models of T cell pathologies [8] including allergic and autoim- Common shorter haplotypes are often subdivisions of the larger mune manifestations [9]. Genetic variation at TNFSF4 has been haplotypesfoundinnon-Africansandsocanbecorrelatedtothese associatedwiththeautoimmunediseasesystemiclupuserythema- [25].Inadmixedpopulations,thegeneticcomponentattributableto tosus (SLE),andother inflammatoryconditions includingathero- the West African ancestral population would equate to a faster sclerosis andischaemic stroke. decayofLD.ThebreakdownofLDisthereforegreaterinAfrican- SLE is the prototypic multi-system autoimmune disorder. Americans, because the majorcomponent(80% ormore) oftheir High-affinity, pathogenic IgG autoantibodies to an array of genome is West-African, compared to Hispanics who have nuclearantigensareahallmarkofpathogenesisandcharacterise componentestimatesbetween4–11%[23,26]. theglobal perturbation of theimmunesystem in SLE.Variation We infer a fine-scale map of the recombination rate and in at least 25 genetic loci with modest effect sizes are thought to location of hotspots within each entire population and in explain the genetic component of SLE [10]. The strong genetic subgroups of interest. We have used principal components (PC)- basis to disease is well-established and has been strengthened by based strategies to adjust for major ancestry before performing a the advent of GWAS, which has corroborated the association of high-resolution association study which utilises typed and proba- immunologically relevant loci with SLE [11–13]. We have bilisticgenotypestomaptheTNFSF4locus.Bysurveyingcommon previously shown single nucleotide polymorphisms (SNPs) in the variants up to 1000 Genomes Phase 1(v3), we aim to identify 59 TNFSF4 region to be associated with lupus in European commoncausalvariationatTNFSF4associatedwithSLE.Cross- families and a case-control cohort [4]. The increased association comparison of associated risk haplotypes across four populations of 59 risk alleles with disease has been replicated in East Asian focuses these analyses.The AAassociation replicates ina smaller populations [14,15], highlighting the genetic similarities at this cohort of AA-Gullah [27]. Our haplotype analyses find informa- locusintheseancestrallydistinctpopulations.MultipleSLErisk- tiverecombinantsinAfrican-AmericansandEuropeanstoresolve associated TNFSF4 variants are also associated with systemic genetic variantsassociated with SLEat thislocus. sclerosis [16], primary Sjo¨gren’s syndrome [17] and myocardial These data are used to perform six case-control association infarction [18,19]. studies and a trans-ancestral mapping experiment using in excess A major obstacle in the identification of disease-specific causal of 17900 subjects. We attempt to define causal variation at variantsatTNFSF4intheEuropeanandEastAsianSLEcohorts TNFSF4 in SLE susceptibility. In a complementary strategy we has been the strong linkage disequilibrium (R2.0.8) exhibited by perform association analysis using TNFSF4 alleles and lupus genotypedTNFSF4alleles,whichhasresultedinahighfrequency phenotypes. We explore the mechanism by which TNFSF4 extended haplotype associated with risk of disease instead of influences perturbation of the immune process in inflammatory delineating causal variations at the locus [4]. It is probable that disease. Finally, we interrogate risk alleles in terms of their migration out of Africa involved many founder effects and influence on transcription factor binding using a bioinformatics bottlenecks to increase haplotype length in East Asian and approach.Theresearchpresentedusestrans-ancestralmappingto European populations [20]. Hispanic and African-American inform thiscomplextrait. PLOSGenetics | www.plosgenetics.org 2 July2013 | Volume 9 | Issue 7 | e1003554 TransAncestralFineMappingofSLELocusTNFSF4 andcommonvariants (SNPsandINDELs)across thelocususing Author Summary MAF.1%intheimputationscaffold.Asdescribedinmethods,we Systemic lupus erythematosus (SLE/lupus) is a complex used a post imputation filter of HWE.0.01 and info .0.7 to disease in which the body’s immune cells cause inflam- include only genotyped and high quality imputed SNPs. The mation in one or more systems to cause the associated estimated concordance between imputed and true genotypes for morbidity. Hormones, the environment and genes are all theSNPs presented inthisstudy is0.95forall cohorts.Thefinal causalcontributorstoSLEandoverthepastseveralyears characteristics of alldatasets are presented in Table1. thegeneticcomponentofSLEhasbeenfirmlyestablished. Severalgeneswhichareregulatorsoftheimmunesystem Bayesian inference of fine-scale map of recombination are associated with disease risk. We have established one of these, the tumour-necrosis family superfamily member rates and hotspot densities at TNFSF4 4(TNFSF4)gene,asalupussusceptibilitygeneinNorthern The European sex-averaged and female-only recombination Europeans. A major obstacle in pinpointing the marker(s) maps generated by deCODE (http://www.decode.com/ atTNFSF4whichbestexplaintheriskofSLEhasbeenthe addendum/), are based on 15,257 and 8,850 directly observed strong correlation (linkage disequilibrium, LD) between recombinations,respectively.Thesemapshavearesolutioneffective adjacent markers across the TNFSF4 region in this down to 10kb and comparing them to the HapMap 3 and 1000 population.Toaddressthis,wehavetypedpolymorphisms Genomespopulation-averagedmaps[30,31],wefounddifferences inseveralpopulationsinadditiontotheEuropeangroups. attheTNFSF4locus.Thus,weestimatedbackgroundrecombina- The mixedancestry ofthesepopulations givesadifferent tion rates in AA, East Asians, Europeans and Hispanics using a LD pattern than that found in Europeans, presenting a Bayesiancomposite-likelihoodmethod.Theinclusionofahotspot method of pinpointing the section of the TNFSF4 region model allowed sampling of hotspots from the Markov chain and which results in SLE susceptibility. The Non-European inference of mean posterior hotspot densities from a threshold populations have allowed identification of a polymor- upwards of 0.25, giving a detection power of 50% and a false- phism likely to regulate expression of TNFSF4 to increase susceptibility to SLE. discoveryrateof4%[32].InAsians,EuropeansandHispanicsthe bulk of the recombination occurs in less than 5% of sequence (Figure1andFigure2).Anexceptiontothispatternisfoundin the African-American cohort, with increased recombination rate Results and higher density and proportion of hotspots across the locus To delineate the causal variation at TNFSF4, we genotyped (Figure1,Figure2).Inallpopulations,peakrecombinationisat SNPs in a 200Kb section of chromosome 1q25.1 encompassing the 59 boundary of the TNFSF4 gene and approximately 120kb TNFSF4 (23.6kb) and the 59 region (150 kb). Population into the 59 region. A difference in African-Americans is that stratification bias and effects due to admixture were addressed recombinationextends30kbfromtheTNFSF4geneboundaryinto using the approach of Namjou and colleagues [28]. We also the59region,whilstthereisnegligiblerecombinationinthissection genotyped 347 SNPs used by Halder [29] to correct for major in the other populations (Figure 1) and this is compatible with ancestry in each population as identified by a PCA-based increasedcomplexityofthegenomicregioninAfrican-Americans. approach (Supplementary Figure S1). As outlined in methods, SNPsandindividualsthatfailedqualitycontrolwerefiltered;pre- Single marker association of 59 TNFSF4 SNPs with SLE and post- imputation SNP counts and a description of the Theassociationdatapresentedareformarkersafterimputation component sample setsispresented inTable1. usingthe1000GenomesphaseIintegratedvariantsetv3(March To directly compare genotyped SNPs we used the phased 2012,NCBIbuild37)[30].TheTNFSF4locusiswellestablishedin chromosomes of1000Genomesphase I integrated variant setv3 SLE therefore we have presented uncorrected nominal p-values (March 2012, NCBI build37) (The 1000 Genomes Project forvariants.InEastAsians,EuropeansandHispanicsmanystrong Consortium) [30] and IMPUTE v2.2, together with second associations (P 1028,10216) at TNFSF4 are detected. Multiple u reference sets defined in Table 1. We imputed missing data susceptibility alleles inthe TNFSF4 59regionare overrepresented Table1. Populationdemographics and imputationreference data forSLE-control cohorts post QCfiltering. European EastAsian Hispanic AA-Gullah Cases Controls TOTAL Cases Controls TOTAL Cases Controls TOTAL Cases Controls TOTAL Males 344 1151 1495 167 225 392 119 73 192 136 593 729 Females 3088 2489 5577 1333 1171 2507 1229 644 1872 1541 1341 2882 Unknown 3 236 239 TOTAL 3432 3640 7072 1500 1396 2896 1348 717 2065 1680 2170 3850 SNPS(TYPED) 89 89 89 65 65 65 51 51 51 88 88 88 SNPS(ALL) 244 244 244 450 450 450 460 460 460 393 393 393 Imputationreference1 1000G 1000G 1000G 1000G 1000G 1000G 1000G 1000G 1000G 1000G 1000G 1000G Imputationreference2 OMNI-QUAD UK-CanadianGWAS Numbersafterfilteringforduplicates,FDRs,HWE,missingnessandmajorancestry.postSNPswithINFOscores,0.7excluded,SNPSwithHWE,0.01excluded. doi:10.1371/journal.pgen.1003554.t001 PLOSGenetics | www.plosgenetics.org 3 July2013 | Volume 9 | Issue 7 | e1003554 TransAncestralFineMappingofSLELocusTNFSF4 Figure1.FinescalemapsofrecombinationrateinferredfromEastAsian,European,HispanicandAfrican-Americancontrolphased chromosomes.1568randomlyassignedchromosomesfromeachgroupweretestedusingRhomap,fromtheLDHAT2.0package.Thefine-scale mapofrecombinationrate(4Ner/kb)wasinferredacross200kbofchromosome1q25encompassingTNFSF4geneandextended59and39regions. doi:10.1371/journal.pgen.1003554.g001 inSLEcases(Table2,Figure2).Intermsofsinglemarkers,best associated in African-Americans and the 59 association replicates evidenceofassociationwithdiseaseinEuropeansisobservedwith in a small cohort of AA-Gullah (Supplementary Table S1), rs2205960-T, 10kb 59 from the TNFSF4 gene (P=5.61610215, underpinning thisgeneas a global SLEsusceptibility gene. OR=1.34(95%CI1.25–1.44)).TheTalleleofrs2205960alsohas In African-Americans, the best evidence for the 59 SNP strongest association with Hispanic SLE (P=1.7610210, association with disease are from rs1234317-T (P=2.2861025, OR=1.65 (95% 1.42–1.91)). In Europeans, an additional 15 OR=1.4 (95%CI 1.25–1.56)) and rs2205960-T (P=7.261025, SNPs reach genome-wide significance (P,561028) most of these OR=1.48 (95%CI 1.22–1.67)) and rs1234314 –G riskallelesalsoreachthislevelofsignificanceintheEastAsianand (P=3.1161025,OR=1.22(95%CI1.13–1.32)).Thereisatrend Hispaniccohorts(Table2).Several59riskallelesassociatedwith for under-representation of the minor alleles of rs1234314-C, disease in East Asians, Europeans and Hispanics are also rs1234315-C,rs844642-G,rs844644-A,rs2795288-Tandrs844654- PLOSGenetics | www.plosgenetics.org 4 July2013 | Volume 9 | Issue 7 | e1003554 TransAncestralFineMappingofSLELocusTNFSF4 Figure 2. Single marker association atTNFSF4locus inA. East Asian,B. European,C. Hispanic,D. African-American SLE-control populations.Thestrengthoftheassociation(-log10uncorrectedP)ofmarkersacross240kbofchromosome1q25.ThisregionalplotdepictsTNFSF4 associationwithSLEversuschromosomalposition(kb)inEastAsians,Europeans,HispanicsandAfrican-Americanpopulations.Themostassociated variantsineachgrouparelabelled,asisthebest-associatedmeta-analysisSNPrs2205960.Markersarecolour-codedfortheircorrelationcoefficient (r2)valuesaccordingtothelegend. doi:10.1371/journal.pgen.1003554.g002 A in SLE cases resulting in a flipped OR for these variants sameimputationanalysisandsubjecttothesameQCastheSNPs (Table2). and probabilistic genotypes incorporated into our association analyses. We identify a deletion at rs200818062 [-/G] to be AssociationofintragenicTNFSF4singlemarkerswithSLE associated with SLE in all groups tested. This indel is located Examining the genetic association between SNPs within the 22.4 kbfromthestartsiteofthecommontranscript(Transcript1) TNFSF4 gene and SLE we identify association of rs1234313-G, of TNFSF4 and is in strong LD with (R2.0.8) rs1234317 and within intron1, with SLE in Asians (P=4.3761028, OR=1.38 rs2205960. (95%CI 1.32–1.44)), and Europeans (P=1.1161025, OR=1.15(95% CI 1.11–1.27). In both cohorts rs1234313-G is Best evidence meta-analysis partitioned from other associated SNPs by a recombination We used a logistic regression model fitted with an interaction hotspot at the TNFSF4- 59 boundary. However, correlation term (effect) in the R statistical package to investigate cross-study coefficient R2 values between this marker and risk-associated 59 heterogeneity. P-values for individual associated SNPs were variants suggest strong correlation. We identify under represen- generated using a likelihood-ratio test. We found no evidence of tationofrs10798265-AinAfrican-AmericanSLE(P=9.2461025, heterogeneityforthekeyrisk-haplotype-taggingcommonvariants 0.84(95%CI 0.78–0.9)). There is suggestion of additional modest which span the locus. Our null hypothesis - that all studies were association signals (P,1024) from a series of SNPs located at the evaluating the same effect size- held true for key variants TNFSF4-39UTRboundary inthesame cohort. associated with riskofSLE. We combined the association data for variants across the 59 Imputation of typed bi-allelic indels TNFSF4regioninEastAsians,Europeans,HispanicsandAfrican- Imputation gave 257 common (.1% MAF) bi-allelic indels at Americans, to more powerfully estimate the true effect size theTNFSF4locus,mostlyneutral.Theindelswereincludedinthe (Table 3). The average effect size across all datasets was PLOSGenetics | www.plosgenetics.org 5 July2013 | Volume 9 | Issue 7 | e1003554 TransAncestralFineMappingofSLELocusTNFSF4 5–0.81) 8–0.84) –1.63) 2–1.69) 51–1.88) 62–0.90) 61–0.88) 2–0.89) 9–1.66) 5–0.80) 42–1.70) 5–1.61) 8–1.65) 1–1.57) 8–1.54) 3–1.04) 54–0.8) 1–0.74) 7–1.64) 37–1.64) 37–1.64) 2–1.58) 38–1.64) 7–1.56) 37–1.64) 38–1.64) 8–1.64) 8–1.66) 34–1.60) 34–1.60) 37–1.63) 33–1.59) 33–1.59) 48–1.61) Hisp 0.68(0.5 0.71(0.5 1.5(1.36 1.56(1.4 1.69(1. 0.76(0. 0.74(0. 0.75(0.6 1.53(1.3 0.67(0.5 1.56(1. 1.48(1.3 1.51(1.3 1.44(1.3 1.41(1.2 0.79(0.5 0.67(0. 0.67(0.6 1.51(1.3 1.50(1. 1.50(1. 1.45(1.3 1.51(1. 1.41(1.2 1.51(1. 1.51(1. 1.51(1.3 1.52(1.3 1.47(1. 1.47(1. 1.50(1. 1.46(1. 1.46(1. 1.48(1.3 4) 0) 1) 0) 5) 5) 5) 5) 5) 5) 6) 26) 5) 6) 6) 87) 88) 34) 40) 1.3 0.9 0.9 91) 36) 85) 1.3 26) 30) 35) 24) 92) 0.8 83) 35) 1.3 1.3 35) 1.3 28) 1.3 1.3 6) 33) 1.2 –1. 1.3 1.2 1.2 36) Eur 0.83(0.78–0. 0.82(0.76–0. 1.27(1.20–1. 1.33(1.26–1. 1.26(1.18– 0.84(0.77– 0.84(0.78– 0.84(0.78–0. 1.29(1.22–1. 0.81(0.78–0. 1.23(1.17– 1.19(1.13–1. 1.23(1.17–1. 1.28(1.22–1. 1.18(1.11–1. 0.85(0.79–0. 0.81(0.78– 0.78(0.74–0. 1.28(1.21–1. 1.28(1.21– 1.28(1.21– 1.29(1.22–1. 1.28(1.21– 1.21(1.14–1. 1.28(1.21– 1.28(1.21– 1.2(1.13–1.2 1.26(1.19–1. 1.19(1.13– 1.197(1.14 1.28(1.21– 1.19(1.13– 1.19(1.13– 1.28(1.21–1. CI) As 0.72(0.67–0.87) 0.72(0.68–0.78) 1.37(1.26–1.48) 1.43(1.32–1.54) 1.55(1.40–1.69) 0.71(0.66–0.78) 0.71(0.67–0.77) 0.71(0.67–0.77) 1.37(1.26–1.48) 0.73(0.68–0.79) 1.37(1.26–1.48) 1.39(1.29–1.50) 1.37(1.26–1.48) 1.33(1.23–1.43) 1.38(1.28–1.48) 0.73(0.68–0.81) 0.73(0.68–0.79) 0.74(0.68–0.81) 1.36(1.25–1.47) 1.36(1.25–1.47) 1.34(1.23–1.45) 1.34(1.24–1.44) 1.34(1.23–1.45) 1.40(1.29–1.52) 1.35(1.24–1.46) 1.37(1.26–1.48) 1.43(1.31–1.55) 1.34(1.23–1.45) 1.377(1.27–1.49) 1.38(1.27–1.48) 1.36(1.25–1.47) 1.37(1.26–1.48) 1.37(1.26–1.48) 1.36(1.25–1.47) ohorts. OddsRatio(95% AAG 0.82(0.76–0.88) 1.02(0.91–1.13) 1.4(1.25–1.56) 1.48(1.2–1.67) 1.45(1.28–1.63) 1.03(0.90–1.16) 1.07(0.93–1.22) 1.00(0.88–1.13) 1.44(1.23–1.65) 0.98(0.89–1.07) 1.38(1.22–1.54) 1.00(0.91–1.11) 1.29(1.15–1.43) 1.05(0.95–1.15) 1.02(0.92–1.12) 0.98(0.91–1.05) 1.02(0.93–1.12) 0.8(0.72–0.90) 1.26(1.12–1.39) 1.26(1.12–1.39) 1.25(1.12–1.39) 1.04(0.94–1.14) 1.26(1.12–1.39) 0.95(0.85–1.06) 1.26(1.12–1.39) 1.27(1.13–1.40) 1.17(1.05–1.29) 1.30(1.10–1.50) 1.18(1.06–1.3) 1.17(1.057–1.29) 1.27(1.14–1.41) 1.18(1.06–1.30) 1.18(1.06–1.30) 1.28(1.15–1.42) (Hisp)SLE-controlc EurHisp 2299662.43103.6710 22107662.91103.0610 22119661.77103.6510 221510665.61101.7010 2298662.54103.3810 2274661.09101.7410 2275661.12102.6210 2275661.04103.0310 221210662.48104.7110 22119669.2101.8510 221010661.7610310 2289669.40103.8210 221010661.79108.410 22138662.59103.0710 2267661.35102.0810 2662.08100.07 22119666.05102.3610 22129665.87106.9110 22129663.83101.2310 22129662.69101.4110 22129662.68101.4110 22138662.22102.9310 22129662.89101.1910 2276661.86102.9210 22129664.53101.2510 22129664.91101.210 22810669.21108.7710 22109664.36103.7510 2289666.04104.1910 2289666.08104.1710 22129664.78101.5110 2289667.82108.9410 2289667.8108.9210 22129662.87107.3710 anic e 211 210 28 210 290 2100 2110 211 28 210 280 29 28 28 210 29 280 28 280 270 290 28 280 270 29 290 280 280 280 2110 210 dHisp p-valu As 65.8410 61.7810 61.8510 61.1810 65.451 62.091 63.981 62.5610 61.3810 65.6210 61.271 61.3610 62.1110 65.1710 66.0310 61.4310 69.471 63.0810 63.301 61.461 64.581 68.2810 61.881 63.161 65.4310 65.471 64.791 61.301 61.301 65.841 61.7810 n d an(Eur)a Unadjuste AAG 2563.1110 0.7 2562.2810 2567.2010 2562.8510 0.65 0.35 0.91 2466.1310 0.6 2566.8210 0.89 2463.0510 0.34 0.68524 0.74281 0.7133 2361.2810 2468.9210 2468.1210 2469.5310 0.47 2469.6910 0.37 2469.0110 2465.9310 0.01 236910 2367.2910 2368.7710 2465.8210 2366.5410 2366.5510 2463.9110 e p o B s),Eur AA/A C/G C/T T/C T/G 7D/I G/T 8G/A A/C A/G T/A 8T/C T/G G/T G/A C/A A/G 9A/T C/A C/T 9T/C 9A/G C/T 9T/C T/A 9T/A T/C T/C A/G 7C/T 7C/T T/C 7T/C 7T/G G/A A 8 4 9 7 1 4 3 9 8 2 7 9 5 4 8 9 2 2 4 2 6 2 3 6 9 3 3 3 3 3 Asian( Hisp 0.42/0.4 0.36/0.4 0.38/0.2 0.37/0.2 0.24/0. 0.34/0. 0.33/0. 0.33/0.4 0.38/0.2 0.42/0.4 0.38/0. 0.46/0.3 0.39/0.2 0.44/0.3 0.5/0.42 0.09/0.1 0.42/0. 0.29/0.3 0.39/0.2 0.39/0. 0.39/0. 0.43/0.3 0.39/0. 0.45/0.3 0.39/0. 0.39/0. 0.46/0.3 0.39/0.2 0.46/0. 0.46/0. 0.39/0. 0.46/0. 0.46/0. 0.39/0.3 3 7 3 7 3 3 3 4 4 4 4 4 4 4 SinglemarkerassociationresultsforEastTable2. F_AA/F_ABMarkerCoordinate AAGAsEur rs1234314173.17740.31/0.360.38/0.70.43/0.43 rs1234315173.17850.2/0.20.39/0.470.47/0.48 rs1234317173.18780.11/0.080.32/0.250.31/0.26 rs2205960173.19150.07/0.050.32/0.250.27/0.22 rs200818062173.19890.1/0.070.26/0.20.27/0.2 rs6673550173.19910.16/0.160.48/0.40.45/0.5 rs844642173.20780.11/0.120.41/0.490.43/0.4 rs844644173.20950.16/0.160.41/0.490.43/0.47 rs12039904173.21230.06/0.040.32/0.250.28/0.23 rs2795288173.21390.47/0.480.41/0.490.41/0.47 rs4916315173.21540.12/0.090.31/0.250.28/0.2 rs1012507173.21950.28/0.280.34/0.270.38/0.34 rs35086785173.22020.13/0.110.31/0.250.28/0.23 rs844648173.22430.29/0.280.42/0.360.33/0.28 rs844649173.22510.33/0.320.44/0.360.43/0.39 rs844651173.23150.15/0.150.49/0.420.43/0.47 rs1099447173.23280.47/0.470.41/0.490.42/0.4 rs844654173.23610.12/0.150.25/0.310.23/0.28 rs10489265173.23670.14/0.120.31/0.250.29/0.23 rs34313362173.24070.14/0.120.31/0.250.29/0.2 rs10912576173.24190.14/0.120.31/0.250.29/0.2 rs844663173.24360.28/0.270.42/0.350.33/0.28 rs12403570173.24440.14/0.120.31/0.250.28/0.2 rs12049190173.24730.29/0.30.33/0.260.38/0.33 rs35634597173.24820.14/0.120.31/0.250.29/0.2 rs67638449173.24930.14/0.120.31/0.250.29/0.2 rs12750070173.24980.19/0.170.33/0.250.39/0.34 rs12405577173.25000.07/0.050.32/0.260.29/0.24 rs6697570173.25410.19/0.160.33/0.260.38/0.3 rs12143114173.25450.2/0.170.33/0.260.38/0.3 rs35691278173.25510.14/0.110.31/0.250.29/0.2 rs11802979173.25630.19/0.160.32/0.250.38/0.3 rs10912579173.25630.19/0.160.32/0.250.38/0.3 rs10912580173.25660.14/0.110.31/0.250.29/0.24 Variantsinboldareimputedusing1000Genomesphase1(v3).doi:10.1371/journal.pgen.1003554.t002 PLOSGenetics | www.plosgenetics.org 6 July2013 | Volume 9 | Issue 7 | e1003554 TransAncestralFineMappingofSLELocusTNFSF4 or 4 1 3 4 3 2 1 rs 0, 14 596 3 0 4 2 3 2 120 rs rs96 23 23 23 on pValuers2205+ 1 64.5710 0.12 1 0.02 0.14 0.13 0.12 0.13 0.10 0.10 0.09 0.08 0.03 0.07 0.01 0.03 0.13 0.11 0.16 0.17 0.15 66.6110 0.10 0.10 69.8610 0.02 0.05 0.04 0.08 0.06 0.12 0.08 0.37 ditioned n o c n, o ati ci o oned SNPass conditi34314 214 28 210 29 29 29 29 29 29 29 29 29 28 29 28 27 26 26 24 24 25 25 24 24 25 23 24 23 24 23 23 23 nominal pValuers12on 64.1210 62.5710 62.3110 1 62.6410 61.2510 61.6710 61.9510 62.6310 62.6210 64.4810 64.8210 65.1410 61.1610 63.4910 61.4110 62.7410 66.6810 61.8310 61.0510 61.3410 65.2110 64.8210 61.8610 61.8610 67.7410 64.7810 66.0910 63.5310 62.2810 67.5110 67.9110 67.2910 0.06 eforthe u al v P d e ail o-t w oned andt pValueconditirs2205960on 1 0.04 0.12 2763.8110 0.01 0.10 0.08 0.07 0.09 0.06 0.06 0.05 0.05 0.02 0.04 2367.0910 0.03 0.02 0.01 0.24 0.25 0.16 2567.7410 0.14 0.13 2565.8010 2461.1410 0.17 2466.610 0.16 2361.7910 0.04 2363.3410 0.03 uencies(FREQ1) q e 4SNPs. pValue 23461.7110 22861.1610 22862.0610 22866.5210 22868.8510 22869.3910 22761.3610 22761.9710 22762.2010 22762.2210 22765.4610 22765.6510 22765.9410 22661.7110 22663.8910 22664.5610 22261.2410 22261.7010 22261.7410 22161.1310 22161.5210 22162.0010 22162.6010 22163.6910 22163.7010 22163.7410 21961.4310 21764.0310 21764.4710 21663.3310 21669.5910 21561.2710 21562.6510 21163.0210 udingallelefr F cl S n eforTNF WEIGHT 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 17096 ysisresultsi u al Pval SE 0.014 0.066 0.097 0.108 0.125 0.137 0.067 0.070 0.070 0.074 0.070 0.038 0.096 0.067 0.067 0.067 0.081 0.042 0.043 0.068 0.046 0.090 0.067 0.082 0.082 0.063 0.084 0.044 0.118 0.078 0.044 0.154 0.082 0.010 eta-an m ciation FREQ 0.549 0.757 0.775 0.396 0.615 0.637 0.755 0.240 0.247 0.764 0.247 0.663 0.767 0.247 0.245 0.860 0.684 0.396 0.323 0.248 0.420 0.779 0.755 0.688 0.688 0.757 0.249 0.661 0.375 0.684 0.672 0.339 0.314 0.547 mnslist o u ass AB col the AA/ G/T C/T C/T C/G C/T C/T C/T T/C A/G C/T A/T G/A C/T T/G A/C I/D C/T T/C T/C T/C T/C C/T T/A G/T C/T T/A T/C G/T A/G T/A C/A A/C T/G G/A extsix of n e videncemeta-analysis COORDINATE(Mb) 173.1915 173.1878 173.2123 173.1774 173.2154 173.2493 173.2407 173.2367 173.2566 173.2551 173.2482 173.2419 173.2444 173.2202 173.2361 173.1988 173.2500 173.2243 173.2436 173.2541 173.2545 173.2498 173.2139 173.2563 173.2563 173.2328 173.1785 173.2195 173.2078 173.2473 173.2095 173.2251 173.1991 173.1915 nslistSNPcharacteristics,thrs1234314rs2205960.+gen.1003554.t003 Beste AME ecolumonbothournal.p Table3. MARKERN rs2205960 rs1234317 rs12039904 rs1234314 rs4916315 rs67638449 rs10912576 rs34313362 rs10912580 rs35691278 rs35634597 rs12046550 rs12403570 rs35086785 rs10489265 rs200818062 rs12405577 rs844649 rs844663 rs6697570 rs12143114 rs12750070 rs2795288 rs10912579 rs11802979 rs844654 rs1234315 rs1012507 rs844642 rs12049190 rs844644 rs844651 rs6673550 rs1099447 Thefirstthreconditioneddoi:10.1371/j PLOSGenetics | www.plosgenetics.org 7 July2013 | Volume 9 | Issue 7 | e1003554 TransAncestralFineMappingofSLELocusTNFSF4 Figure3.LDplotsatTNFSF4locusinfourpopulations.Thissectionofchromosome1q25.1encompassestheTNFSF4geneandupstream regionasdefinedbycustomalgorithminHaploview4.2.ThemeasureofLDwasusedtodepict57SNPscommontoallcohorts,postQCand1000 genomes imputation. The pair-wise correlations between TNFSF4 markers is illustrated in these plots by the correlation coefficient R2(where r2=0=no correlation, white; 0,R2,1, gradations of grey; R2=1=complete correlation, black). The TNFSF4 gene is positioned above the plots relativetohaplotypeblocks(blacktriangles)andgreyticksindicateSNPlocationstoscale. doi:10.1371/journal.pgen.1003554.g003 computedusinginversevarianceweightingofeachstudy.Wefind Conditional regression analysis of 59 single-markers the 59 association of TNFSF4 with SLE greatly reinforced. As expected, our 59 association data suggest pairwise LD rs2205960-T, the most associated allele in Europeans and betweenmarkersisweakestinAfricanAmericansandstrongestin Hispanics, (P=1.71610234, OR=1.43[1.26–1.60]), and Asians.Inordertoestablish whetherthesignalsidentifiedbyour rs1234317-T (P=1.16610228, OR=1.38[1.24–1.54]) have the trans-ancestralfine-mappingexperimentrepresentcausalvariants, strongest combined association with disease (Table 3). These independent risk factors, or if we have simply found surrogate adjacent markers are separated by 3 kb of chromosome 1. Allele markers strongly correlated with causal variants, we conditioned frequencies for rs2205960 for 1000 Genomes populations are theassociationdatafromeachpopulationwiththemarkerwhich presented insupplementary data. represented thebest evidenceofassociation. PLOSGenetics | www.plosgenetics.org 8 July2013 | Volume 9 | Issue 7 | e1003554 TransAncestralFineMappingofSLELocusTNFSF4 Inallpopulations,rs2205960-T,arisk-haplotypetagSNPwith 0 6 highp-valueandeffectsize(Table3,Figure3)isassociatedwith 9 05 SLE; a similar trend is illustrated by the adjacent marker rs22 0.009 0.94 21 trhs1e2s3i4gn31al7a-Ttr.s1C2o3n4d3i1ti7onisinlogstoanftersr1c2o3n4d3i1ti7onoinrgrsf2o2r0r5s29260059w6e0,fianndd 7 this is consistent for across populations (Table 4). If the reverse 1 Hispanic rs12343 0.005 21 0.010 arEsn1ua2rlo3yp4sie3sa1n7iss,atpnhederrfHeoriismspeardnesicidasnu(Padl=wa9se6so1cc0ioa2nti4doAinSti,oPant,r1os20n2204t5hE9Ue6,0Ppr=iens0e.An0c1sei5aHnois)f., n/ In all apart from the AA group, conditioning on rs1234317 or a 4 Amerindi p-value rs123431 21 0.007 2463.5610 rrss22220055996600,lres1a2ve3s43a17reasniddurasl12s3ig4n3a1l4aitnrtsh1e2s3e4a3n1a4l.ysWes.e included Conditional analysis of meta-analysis data 960 m. We find conditioning on the presence of rs1234317 or 05 dig rs2205960, association of intron 1 markers tested for all groups rs22 0.038 0.57 21 para (iTs alobslte, 2c)o.nWfiremailnsogctohnedseitioansedsetchoenmdaertya-atnoal5y9sisraissksocaisastoiocinatdioantas 34317 25610 equentist o(Pn=3rs.28210651906207),alnodcatfeoduantdthereTsiNduFaSlF4a-5ss9obcioautinodnarya.tTrhse12r3ev4e3r1s4e rs12 0.017 21 4.88 dafr (aPn=aly4s.i1s26fo1u0n2d14).inTcrheeasseeddatraesisduugaglestastswocoiaitniodnepeantderns2t2s0ig5n9a6l0s n European p-value rs1234314 21 2568.9810 2864.020 onalmodela arwsn1itd2h3r4sin232c1r04e5ai9ns6eSd0LraEesms.oCocvoiaentdidoiantisosanoncindiagteitofhfneecamtteaTttaN-rdsF2aS2tFa045o9(nT6b0aobctlhoemrs3p1)a2.r3e4d31to4 5960 gadditi Mcoohdoerltlsing the pattern of inheritance in independent rs220 0.013 0.024 21 edusin assIonciagteinonotyopfe-bTaNseFdSFa4nawlyistehs,StLhEe maroedetlshethaadtdbiteisvte/fidtsomthinea5n9t 17 elect models. 3 s rs1234 246410 21 246910 Pvalues Hhaapplloottyyppeesbifurcation of TNFSF4 risk and non-risk groups. Asian p-value rs1234314 21 0.190 0.008 gframework. ioddfieaHngLrtaiaDpfmileodstoyffpoferraossemsasoicgchinapiptfheiocadpaseundhltalaytpioglaonesnt.syooTpcteyoiasp,tbeesedwtteewfroirtcvhoisnuersaaitsclrkiuhsecottefchdoedhibsboerirafetusaerkctdaewostitewoernnde TNFSF4variantsinfour rs1234317rs2205960 0.0880.103 210.084 20.2241 uscovariatewithinaclusterin tTmtdrT(Fhhsi1Nhseeeie2getFaqf3luiSi-lug4noraFui3enecl4ri1aabeig4tn4lriye,(io,lusneanitmreasbh,-iceoe5sahl(f9lLkreemrpbD)ds.lo1a,oo)Tu2lcstast3niohtrc4decoipm3naurrp1rclraoay7relrxoreskaitpaaecmsnsonoddianindrloleldwgunrsfhsgsdtrt2eotioircos2namtht0otahte5nthiewsy9cetpduh6eheniss0ieacedu,ighdnbmrbSraahbebeNsmasoaetpttP-kr.hhaldoTeosodtlsfhcoyowiopercscnearaeitceamthetbtoviiedprocfadaklnrelnniaissinacnetfnkhwsrsatotteihhgstmionh)eeef. 9Conditionalregressionresultsfor5Table4. AAGullah+MarkerA1A2Coordinate p-value rs1234314 2rs1234314GC1731773921 rs1234317TC1731877750.004 rs2205960TG1731914750.001 ConditionalanalysesinSNPTESTv2CaseControl.Continuodoi:10.1371/journal.pgen.1003554.t004 thrAdoCTt(ats(FThhiunifoaoNofseeiprapTdSwntgnFkiphlbichhnucogSso)neal,oaetnrieFreonoynprTfteir4nf-npoefilv5nAiNo-Lleoc4lar)gtlti,maFon.Doh,iyntsnwS-LtpeticknerohhtFoeetoruiloeeshyn4ihn,dwkrcssneaieabaltlsbo-pihoihsrnrknrnAbsyelcaafesoioeciu.npAsfsTtHcraoiusclykniiWoNmnsahrapiedg)ktsct.crFeeynbrpieeaevsheCSopcdiathinasuiFoednifomoonhaptipm4uinnnrrmtcdalo,aaeiotdpspoihrstdlotclitaasalnlynaaaaozybssipndrtpytnsttareao(iyeidgidasSnnnlc.pboontbiguagdieEouvtttcrtepsssytieurhesbsnhtsiappatrr1meghersokfilje2ineieucepdinrc3llonmeeoaofkds4oEpicacrwnntns3uotnearogeclni1unmiosnoss-inp7t,dsrdccntocbauaeataAswrafiinnutlnnnerniasigLtdoardtayidteehaesnweDtiaenioplroiecddh.oasFstinnah2chioftFisst(rs2mgtFhtprioteta0nhauoocirhorn5gtgeotrpoezec9tsumebushype6bieruglneo0bracfeosterS.ttitrrssnoiiaii3ooi4mssaikatnnn)kky,--t, PLOSGenetics | www.plosgenetics.org 9 July2013 | Volume 9 | Issue 7 | e1003554 TransAncestralFineMappingofSLELocusTNFSF4 Figure 4. Haplotype Bifurcation Diagrams ofTNFSF4 andTNFSF4 for Four Populations. Plots are constructed using phased risk non-risk haplotypesfora.EastAsians,b.Europeans,c.Hispanicsandd.African-AmericansandillustratebreakdownofLDwithincreasingdistancesfromacore proximalTNFSF4SNPandareapproximatelytoscale.ThecoreislocatedattheTNFSF4gene-59boundary(blackcircle)andisselectedasthemost proximal59marker,rs1234314,ineachpopulation.Genelocationisdepictedtoscale;wehaveadditionallylabelledeachplottoshowthelocationof rs1234317andrs2205960,thebest-associatedmarkersfromthemeta-analysis. doi:10.1371/journal.pgen.1003554.g004 the TNFSF4-59 boundary in East Asians, Europeans, and population,whilstTNFSF4 isthemostfrequenthaplotypefor OR,1 Hispanics allow for the construction of near-identical haplotype all cohorts testedbut underrepresented in SLEindividuals. blocks including risk and non-risk haplotypes (designated The risk haplotype found in East Asians, Europeans and TNFSF4 and TNFSF4 , respectively)(Figure 3) which HispanicsisfragmentedintheAfrican-Americancohort;themost OR.1 OR,1 extend approx. 80kb into the TNFSF4 59 region. Multiple associated risk haplotype is 11kb (P=2.1261025, OR=1.52). associated risk alleles uniquely tag TNFSF4 , the risk Thishaplotypeblockextendsfromrs1234317tothebi-allelicindel OR.1 haplotype, which is overrepresented in SLE individuals in each rs200818062. Only one allele uniquely tags this haplotype, PLOSGenetics | www.plosgenetics.org 10 July2013 | Volume 9 | Issue 7 | e1003554

Description:
Jennifer A. Kelly12, Javier Martin18, Joan T. Merrill19, Kathy L. Moser12, Timothy B. Niewold20, Lopez-Neyra, Consejo Superior de Investigaciones Cientificas, Granada, . Trans Ancestral Fine Mapping of SLE Locus TNFSF4.
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