Introduction. JA Runstadler 1,HSäilä 2, A Savolainen 2, M Leirisalo-Repo 3, K Aho 4, E Tuomilehto-Wolf 4, J Tuomilehto 4,5 and MF Seldin 1 1

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1 (2003) 4, & 2003 Nture Publishing Group All rights reserved /03 $ Anlysis of MHC region genetics in Finnish ptients with juvenile idiopthic rthritis: evidence for different locusspecific effects in polyrticulr vs pucirticulr subsets nd shred DRB1 epitope JA Runstdler 1,HSäilä 2, A Svolinen 2, M Leirislo-Repo 3, K Aho 4, E Tuomilehto-Wolf 4, J Tuomilehto 4,5 nd MF Seldin 1 1 Rowe Progrm in Humn Genetics nd Moleculr Medicine, Deprtments of Biologicl Chemistry nd Medicine, University of Cliforni, Dvis, USA; 2 Rheumtism Foundtion Hospitl, Heinol, Finlnd; 3 Division of Rheumtology, Deprtment of Medicine, Helsinki University Centrl Hospitl, Helsinki, Finlnd; 4 Ntionl Public Helth Institute, Helsinki, Finlnd; 5 Deprtment of Public Helth, University of Helsinki, Helsinki, Finlnd This study used Finnish juvenile idiopthic rthritis (JIA) probnds with pucirticulr nd rheumtoid fctor (RF) negtive polyrticulr subtypes of JIA to further define the genetic susceptibility to JIA. We exmined 16 mrkers spnning n 18 cm region of chromosome 6 encompssing the MHC nd surrounding genomic region in set of 235 Finnish JIA nucler fmilies nd 639 Finnish control individuls. Anlysis by cse/control ssocition nd trnsmission disequilibrium test (TDT) methods ech demonstrted strong evidence for susceptibility locus ner the D6S2447 microstellite (Po10 6 for both methods) tht is flnked by DQB1 nd DRB1. Anlysis of the DRB1 locus suggested tht DRB1*0801 nd DRB1*1101 rther thn DQA1 or other HLA lleles my be responsible for conferring susceptibility to disese. These findings re consistent with the most compelling results of previous reports on HLA ssocitions nd suggest JIA DRB1 shred epitope encompssing criticl mino-cid residues in the third hypervrible region of this molecule. Most importntly, in pucirticulr ptients, the strong ssocition does not extend to proximl mrkers s it does in polyrticulr ptients (Po ). Anlysis strongly suggests tht the difference is becuse of dditionl JIA susceptibility loci within the MHC being present in polyrticulr RF negtive ptients. (2003) 4, doi: /sj.gene Keywords: JIA; JRA; MHC; TDT; linkge disequilibrium Introduction Juvenile idiopthic rthritis (JIA), formerly clled juvenile rheumtoid rthritis (JRA) or juvenile chronic rthritis (JCA), is defined s rthritis of unknown cuse of t lest 6 weeks durtion strting before the ptient s 16th birthdy. 1 Severl subgroups of JIA re recognized nd they differ in clinicl mnifesttions, prognosis, specific utoimmune fetures, nd genetic determinnts. Ethnic differences exist ccording to subgroups nd gender. In Western societies, the nnul incidence of JIA is 10 20/ in the peditric popultion nd the prevlence is bout 1/ Pucirticulr types of onset predominte, comprising bout 60% of ptients. The proportion of rheumtoid fctor (RF) negtive polyrthritis is bout 30%. JIA is complex genetic disese for which there is evidence supporting both genetic nd environmentl effects. It is nonetheless commonly believed tht the risk Correspondence: Dr JA Runstdler, Room 4439, Tupper Hll, Deprtment of Biologicl Chemistry nd Medicine, One Shields Avenue, University of Cliforni, Dvis, Dvis, CA 95616, USA. E-mil: jrunstdler@ucdvis.edu to sibling with JIA is not prticulrly strong, s evidenced by the rrity of reported extended multiplex fmilies. 3 Yet our recent dt provided evidence tht the genetic component in JIA is more mrked thn previously believed, 4 certinly much higher thn in dult rheumtoid rthritis (RA). The first HLA ssocitions with JIA were reported in 1979/80. 5,6 No specific locus hs shown direct, definitive involvement in the pthogenesis of JIA. However, over 50 studies comprising different popultion groups hve shown some consistent ssocition of HLA loci with specific subtypes of disese (see reviews in Glss nd Ginnini 3, Albert et l, 7 Nepom 8, Howrd et l 9 nd Forre nd Smerdel 10 ). The most convincing genetic ssocitions involve the DRB1, DQA1, DQB1, nd DPB1 HLA genes on chromosome 6. However, there is disgreement over the importnce nd implictions of specific HLA ssocitions in JIA. The frequency of HLA lleles vries widely between popultions nd differences in HLA typing methodologies cn mke HLA comprisons inexct. Associtions in Europen derived popultions hve been shown with pucirticulr (DR5(11), DR8, nd DP2(DPB1*0201)); RF negtive polyrticulr JA (DR5(11),

2 DR8, nd DP3(DPB1*0301)); nd RF positive polyrticulr JA (DR4). In limited study, Africn-Americns nd Mexicn-Americns hd similr ssocitions to Europens in contrst to wht hs been reported for other ethnic groups (for exmple, Asins). 11 Some evidence exists tht second DR or DQ gene my increse risk. For exmple, the combintion of DR5/DR8 ws found more often thn expected in ptients. 12 Aside from HLA- A2, the only other clss I locus to be identified is HLA- B27, which is prevlent in older pucirticulr ptients whose disese progresses to spondylorthropthy nd in other JIA ptients tht my crry poor prognosis HLA-DRB1*04, 07, DR2, nd to less significnt extent, 03 hve been seen s modertely protective. 16,17 In ddition, there is evidence for promoter polymorphisms in the DQA1 locus tht re ssocited with JIA in Germn popultion of ptients. 18 However, other studies, 17 while confirming DR findings, found no evidence for DQ predisposition. Mny of the DR-DQ hplotypes re complicted by linkge disequilibrium (LD) so it is difficult to tell which is the primry ssocition, 19 but there is some evidence tht this ssocition is becuse of DRB1-08*. 20 In fct, nlysis of the MHC region of chromosome 6 is complicted by extended LD within this segment. 21 Aside from the current study, only one other mrker ssocition study hs been performed tht exmined multiple mrkers in the MHC region in ddition to specific HLA genes. Exmintion of 177 Germn erly onset pucirticulr JIA ptients nd 261 Cucsin Germn controls sw no ssocition of seven MHC region microstellites (D6S105, D6S510, TNF, TNFc, TNFd, nd TNFe, HSP) nd no greter ssocition of D6S2447 thn with the HLA lleles DQA1*0501 nd DQB1* Moroldo et l 23 were the first group in 1998 to exmine previously typed ptients nd their prents (101 Cucsin fmilies) with the trnsmission disequilibrium test (TDT). Their TDT results estblished fmily-bsed ssocitions of HLA-A2, B27, B35, DR5, nd DR8 to pucirticulr onset JIA. However, no overll TDT sttistic ws clculted nd no other subgroups were exmined. HLA llele ssocitions hve often been wek in JIA studies nd dependent on the popultion being exmined. Linkge my therefore be implied where none exists, becuse of popultion strtifiction. In ddition, the involvement of other genes within the HLA region hs not been ruled out. This study is the beginning of n ttempt to use n isolted founder popultion (Finnish) to more thoroughly investigte the presence of susceptibility loci on chromosome 6. Founder popultions re more likely to hve fewer susceptibility genes present in initil founders nd to hve lrger regions of LD. These chrcteristics cn mke studies utilizing founder popultion more powerful in their bility to find susceptibility loci. The current Finnish popultion (B5 million) rose from limited number of founders bout yers go. This initil founding popultion hs undergone severl bottlenecks, the most recent being fll to people in the erly 18th century mking it n excellent isolted founder popultion. 24,25 In this study, we used conventionl cse control sttistics s well s the TDT, fmily-bsed ssocition test tht is dependent on linkge nd LD between lleles tested nd the susceptibility locus, to exmine the DRB1 locus nd 16 polymorphic mrkers spnning the MHC. Our study popultion included both pucirticulr onset nd polyrticulr onset RH-negtive probnds of Finnish origin. We lso exmined the hplotypes of mrkers ssocited with disese in n ttempt to clrify the reltion between the two subtypes of disese nd susceptibility to JIA in Finlnd. Results Onset nd ssocition When the ptient groups were subdivided by ge of onset, the ssocitions observed were much stronger for ptients of erly onset (o6 yers) rther thn lte onset (46 yers), lthough ssocition ws present to some extent in lte onset ptients (see Tble 1). Given tht the lte onset group of ptients is reltively smll nd their ssocition my be complicted by the inclusion of different disese subtypes, 1 the observtions here will be limited to the erly onset ptients nd their subclssifiction into pucirticulr nd polyrticulr RF-negtive disese (herefter referred to s polyrticulr). Mrker ssocitions We exmined cse control ssocitions by both llele nd genotype t ech of the 16 mrker loci (Tble 1 nd see Web Tbles nd b [ edu/juvenile_rheumtoid_arthritis.pdf]). Within the 20 cm region covered by these mrkers, there re two ssocited subdivisions. The strongest of these is the region spnned by D6S2446 through MID104 in the proximl region of 6p21.3. For both subtypes of disese, polyrticulr nd pucirticulr, D6S2447 (formerly known s DQCAR) nd D6S2446 (formerly known s DQCAR2) were strongly ssocited (Po10 6 ). The MID108 nd MID104 mrkers tht lie kb distl of D6S2447 were very strongly ssocited with only polyrticulr disese (Po for both genotypic nd llelic ssocition). Continuous ssocition lso extended in polyrticulr ptients to D6S273, microstellite locted 785 kb proximl to D6S2447. A second region of ssocition ws suggested in the segment bounded by mrkers D6S461 nd D6S306 locted kb proximl to D6S2447. However, the ssocitions re mrginl nd primrily confined to the genotype. The strongest ssocition in this region ws in the polyrticulr group of ptients with D6S464 (Po10 2 by both llele nd genotype). Mrker TDT The results of the mrker-by-mrker llele nd genotype ssocitions re mirrored by the results of our locus-bylocus TDT nlysis (Tble 2 nd see Web Tble c [ roweprogrm.ucdvis.edu / Juvenile_Rheumtoid_Arthritis.pdf]). Agin, the strongest ssocitions re with D6S2447 nd D6S2446. However, the number of informtive fmilies tht we were ble to nlyze for the pucirticulr probnds ws reltively smll nd the TDT nlysis for this group only tends to significnce t the P ¼ 0.05 level in comprison with the polyrticulr probnds. Significnt ssocition is chieved for the polyrticulr group of ptients for the D6S2446, D6S2447 (Po10 6 for both), MID108, nd MID104 mrkers 327

3 328 Tble 1 Allele nd genotype ssocition for JIA subtypes t 19 genetic loci cm Kb b A/G c Totl Puci Poly EO totl d EO puci EO poly # Controls : # Ptients 612 : : : : : : 95 D6S461 e A G D6S A G D6S A G D6S A G D6S A G D6S A G TNFc A G D6S A G DRB A o o o o G o o D6S A o o o o o G o o o ND f o D6S A o o o o o G o o o o o MID A G MID A o o o G o o o D6S A G D6S A G D6S A G D6S A G Genetic mp distnce in centimorgns obtined from Mrshfield Genetics ( b Physicl mp distnce in kilobse pirs DNA is dpted from the UC Snt Cruz Genome Browser 22 December 2001 relese ( genome.ucsc.edu/). c Cse control ssocition by llele (A) or genotype (G). d Ptients re divided by subtype nd onset (erly onset (EO)). e For reference to the physicl mp, HLA gene loctions in kb re: HLA-AF32989, HLA-BF34333, HLA-DQA1F36199, HLA-DQB1F36215, HLA-DPB1F f Insufficient number of lleles with totls >5 for w 2 clcultions from collpsing smll columns (see description of Clump lgorithm in Mterils nd methods. (P ¼ nd 0.013, respectively). TDT ssocition is lso extended telomeric in the polyrticulr group to the D6S273 locus (P ¼ 0.023). In the second region showing possible ssocition in the cse control nlysis, the only locus tht remined significnt in the TDT ws D6S464 tht ws most suggestive for the combined erly onset subtypes (P ¼ 0.005). This locus showed mrginl significnce in the TDT for both the pucirticulr nd polyrticulr groups of ptients (P ¼ nd 0.02, respectively). TNFc is billelic microstellite locus tht showed no evidence of ssocition by stndrd cse control methods but showed some evidence using the locus TDT in the combined group (P ¼ 0.009) nd tended to significnce in the subgroups (see Tbles 2 nd 3). For those mrkers tht were significnt by locus TDT, llelic ssocition, nd genotypic ssocition, odds rtios nd their 95% confidence limit were clculted (Tble 4). In ddition, the TDT ws lso performed for ech llele to determine the lleles responsible for the differences in ssocition (see Web Tble d [ For the pucirticulr subgroup, the D6S2446 llele 198 showed the strongest ssocition nd bis in trnsmission to probnds (40% in ptients vs 7.8% in controls; OR ¼ 7.8 [95% CI ], TDT P ¼ 0.008). For the polyrticulr subgroup, strong ssocition nd trnsmission distortion is seen for the D6S2447 llele 113 (36.8% vs 10.9%; OR ¼ 4.7 [95% CI ], TDT Po10 6 ), D6S2446 llele 198 (56.8 vs 7.8%; OR ¼ 15.5 [95% CI ]), MID108 llele 157 (71.5 vs 53.5%; OR ¼ 2.2 [95 CI ], TDT P ¼ 0.002), MID104 llele 126 (83 vs 61.9%; OR ¼ 3.0 [95% CI ], TDT P ¼ 0.012, nd D6S (15.9 vs 9.3%; OR ¼ 1.8 [95% CI ], TDT P ¼ ). Polyrticulr vs pucirticulr The power of the TDT nd ssocition nlyses re decresed in the pucirticulr subgroup becuse of the

4 Tble 2 Trnsmission disequilibrium test for JIA t 19 genetic loci 329 cm kb b Totl Puci Poly EO totl c EO puci EO poly D6S461 d D6S D6S D6S D6S D6S TNFc D6S DRB o o D6S ND e ND e ND e D6S o o o MID MID D6S D6S D6S D6S See footnote of Tble 1. b See footnote b of Tble 1. c Ptients re divided by subtype nd onset (erly onset (EO) or lte onset (LO)). d See footnote e of Tble 1. e Number of lleles with >5 informtive fmilies is insufficient (o2) to clculte the globl TDT. Tble 3 TNFc Alleles with significnt (Po0.05) TDT ssocition with JIA Locus nme Position (kb) Allele Onset b Combined c T/ET d Puci T/ET Poly T/ET TNFc All (99) 137/ (46) 66/ (53) 71/66 EO (66) 86/ (31) 40/ (35) 46/ All (99) 63/ (46) 26/ (53) 37/42 EO (66) 46/ (31) 22/ (35) 24/29 See footnote b of Tble 1. b Onset is listed for both lte nd erly onset combined (All) or for erly onset only (EO). c Allele-specific trnsmission disequilibrium test P-vlue. Number of informtive fmilies is in prentheses. d Number of lleles trnsmitted/ expected trnsmitted to probnds. Cse : control specific numbers for selected ssocited lleles re listed by control, erly onset pucirticulr, nd erly onset polyrticulr s follows: TNFc (165) F 760/1074, 72/102, 3/174. All llele-specific totls cn be viewed on the internet ( smller number of fmilies vilble for nlysis compred with the polyrticulr subgroup. Therefore, it is possible tht the MID108 nd MID104 ssocitions by llele, genotype, nd TDT re strong in polyrticulr probnds reltive to pucirticulr probnds only becuse of the incresed number of probnds nd fmilies exmined. This concern ws ddressed by rndomly smpling n equl number of polyrticulr probnds s the number of pucirticulr probnds. This new dt set ws exmined for cse control ssocition by llele nd by genotype t the MID104 locus. This procedure ws repeted 100 times for the MID104 locus nd we found tht the difference between the pucirticulr subgroup nd polyrticulr group remined (men Po10 5 for both llele nd genotype ssocitions; rnge ¼ ). Hplotype exmintion While the ssocition seen t the D6S2446 nd D6S2447 microstellites is strong, hplotypic ssocition ws lso exmined to provide nother mesure of the extent of the ssocition in the MHC clss II region nd in the D6S464 region. No combintion of djcent two or three mrkers tht did not include one of the mrkers inside of D6S273 MID104 region ws significntly ssocited (P40.05) (dt not shown). Figure 1 shows significnt two locus hplotype ssocitions tht were found mong ll mrkers exmined (Po0.001). No significnt two locus hplotype cse control ssocitions were found in the erly onset pucirticulr subtype. However, the polyrticulr group showed n extended rnge of two locus hplotype ssocitions. Hplotype ssocition included the segment between D6S2447 nd D6S497 nd between s fr distlly s we exmined (D6S1616) nd s fr proximlly s TNFc in combintion with MID104. DRB1 ssocition nd TDT The DRB1 locus ws very strongly ssocited by llele, genotype, nd locus TDT (for polyrticulr Po10 5 nd for pucirticulr Po10 3 for cse control ssocition nd for polyrticulr P ¼ nd for pucirticulr P ¼ for TDT) (Tbles 1 nd 2). The OR nd llelic

5 330 Tble 4 Odds rtios for lleles with significnt (Po0.05) llele nd mrker TDT ssocition with JIA Locus nme Position (kb) Allele Onset b Combined c 95% CI Pucirticulr 95% CI Polyrticulr 95% CI D6S All EO D6S All EO D6S All EO All EO All EO All EO MID All EO All EO MID All EO All EO See footnote b of Tble 1. b Onset is listed for erly onset nd lte onset combined (All) or for erly onset only (EO). c Cse : control specific numbers for selected ssocited lleles re listed by control, erly onset pucirticulr, nd erly onset polyrticulr s follows: D6S464 (208)F100/1070, 15/106, 26/168; D6S2446 (198)F96/1224, 28/70, 83/144; D6S2447 (113)F109/996, 26/100, 66/180; MID108 (157)F601/1224, 63/106, 131/184; MID104 (126)F699/1130, 68/106, 154/186. All llele-specific totls cn be viewed on the internet ( Two Locus Hplotype Assocition D6S 461 D6S 299 D6S 464 D6S 478 D6S 1558 D6S 306 TNFc D6S 273 HLA-DRB1 D6S 2446 D6S 2447 MID 108 MID 104 D6S 497 D6S 291 D6S 1641 D6S 1616 Polyrticulr (p<0.001) Telomeric Centromeric Chromosome 6 25,000,000 bp HLA-A HLA-DRB1 HLA-DPB1 HLA-DQA1 HLA-DQB1 50,000,000 bp Figure 1 Two locus hplotype ssocition. Two locus cse control hplotype ssocitions re present for JIA with polyrticulr disese. For the indicted chromosoml mrkers on ech line, ll combintions of two locus hplotypes were ssocited with disese (Po0.001). In contrst, there were no two locus hplotypes ssocited with disese for the pucirticulr subtype. TDT (Tble 5 nd see Web Tble e [ ucdvis.edu / Juvenile_Rheumtoid_Arthritis.pdf]) indicte tht the DRB1*08 subclss (nd DRB1*0801 in prticulr) is strongly ssocited nd significntly trnsmitted in excess to ptients of both polyrticulr nd pucirticulr subgroups (26.5 nd 35.5% vs 13.5%;

6 Tble 5 DRB1 lleles with significnt (Po0.05) TDT ssocition with JIA 331 Locus nme Position (kb) Allele b Onset c Combined d 95% CI Pucirticulr 95% CI Polyrticulr 95% CI DRB *04 All EO *08 All EO *11 All EO *15 All EO See footnote b of Tble 1. b There were no control hplotypes contining DRB1*12, so the odds rtio cnnot be clculted. c See footnote b of Tble 4. d Cse : control specific numbers for selected ssocited lleles re listed by control, erly onset pucirticulr, nd erly onset polyrticulr s follows: DRB1 (08*)F42/312, 29/104, 63/174; DRB1 (15*)F39/312, 4/104, 3/174. All llele specific totls cn be viewed on the internet ( Tble 6 DRB1 llele mino-cid sequence comprison for the 3rd hypervrible nd surrounding region DRB1* Y W N S Q K D F L E D R R A L V D T Y C R H N Y G V G A L - Q A L - Q K - - G R - - N V L - Q K A I G Q A A V 1201 S I A I - Q A A V Amino-cid position is indicted in reference to strt codon. Dshed line indicte identity to the reference DRB1*0801 sequence. OR ¼ 2.5 [95% CI ] nd 3.6 [95% CI ], TDT P ¼ nd o10 6, respectively). In pucirticulr probnds, the DRB1*11 subclss ws lso ssocited nd trnsmitted in excess (OR ¼ 2.7 [95% CI ], TDT P ¼ 0.034) lthough the frequency of this llele ws lower in the popultion (4.2% DRB1*11 in control subjects vs 13.5% DRB1*08 in control subjects). The DRB1*12 subclss (which is relted to DRB1*11) ws not present in the control. popultion nd, lthough it showed slight trnsmission distortion in polyrticulr ptients, the overll frequency ws very low in this group (1.7% of hplotypes). On the other hnd, DRB1*15 showed very low odds rtio nd ws not trnsmitted s often s expected to the polyrticulr group of ptients (1.7% vs 12.5%; OR ¼ 0.1 [95% CI ], TDT Po0.001 fter correction for compenstory decrese becuse of overrepresented lleles (see mterils nd methods)). DRB1*04, lthough the odds rtio is not significnt, ws trnsmitted less frequently to probnds thn expected in both subgroups (TDT P ¼ nd for pucirticulr nd polyrticulr groups, respectively). DRB1 llele exmintion Subsequent to our results from nlysis of the DRB1 locus, we chose to exmine the sequence of the puttive susceptibility nd protective lleles for DRB1. Tble 6 shows the mino-cid sequence of the third hypervrible region of the DRB1 molecule. It is in this region tht the DRB1*08 nd DRB1*11 subclsses shre nonconservtive phenyllnine substitution. This substitution is the only region tht is unique to these two subclsses within the DRB1 gene coding sequence. In ddition, it is in this region tht the DRB1*15 subclss hs unique nonconservtive lnine substitution. Discussion The current dt re consistent with the Finnish pucirticulr nd polyrticulr ptients shring HLA-DRB1 in common s susceptibility locus. The DRB1*0801 llele ws the most strongly nd uniformly ssocited llele cross both subtypes nd our sequencebsed typing of the locus did not revel ny disesespecific muttions in probnd smples. The fct tht other relted DRB1 lleles re less strongly ssocited with disese (DRB1*11 in pucirticulr ptients) is not inconsistent with this ide. Of potentil importnce, pucirticulr disese ws more strongly ssocited with the DRB1*11 thn polyrticulr disese. The difference between DRB1*08 nd DRB1*11 (DR5 specificity) is unlikely to be sufficient to understnd why the DRB1 polyrticulr disese ssocition ppers to be restricted to DRB1*08. A single

7 332 conservtive mino-cid difference (lnine vs leucine) is present in the criticl third hypervrible region. An lternte explntion is tht dditionl loci tht modify the effects of the DRB1 locus re present nd different on the hplotypes of given DRB1 llele. For exmple, DRB1*08 nd DRB1*11 my be sufficient for the HLA contribution to pucirticulr disese wheres for polyrticulr disese dditionl modifying loci, in prticulr DRB1*08 hplotype my lso be required. There is lso evidence for protective effect of specific DRB1 lleles. After correction for the incresed frequency of DRB1*08, DRB1*15 ws found t smller frequency nd ws trnsmitted less often to probnds in polyrticulr JIA. It is possible tht protective lleles t the DRB1 locus (nd other HLA loci) brogte the synergistic effects of other susceptibility loci. It my be importnt tht DRB1*15 does hve nonconservtive mino-cid substitution within the third hypervrible region (see Tble 6), which my explin some of its protective effects. DRB1*15 is unique in this respect becuse no other subclss contins unique nonconservtive chnges within the third hypervrible region. This region of the DRB1 molecule is involved in the binding of ntigen nd recognition by the trimoleculr complex. Strong evidence exists for the ssocition of the DRB1 locus with other immune-medited diseses such s RA nd type I dibetes. As in RA where the DRB1*04 subclss of lleles contins strongly ssocited shred epitope, 26 we hve proposed shred epitope unique to JIA pucirticulr nd polyrticulr disese. This epitope is different from the RA shred epitope but contins unique nonconservtive substitution (phenyllnine) different from ny other DRB1 llele. This epitope my be criticl to the formtion of the trimoleculr complex. It should be noted tht in ddition to DRB1, there re lrge number of other genes with demonstrted or proposed immune-medited ctivity tht re in strong LD in the MHC region with the locus where we hve shown strong ssocition for both pucirticulr nd polyrticulr disese (brcketed by the TNFc microstellite proximlly nd the MID104 locus distlly). This region includes genes involved in immune signling nd inflmmtion (TNF, LTB, AIF1, nd HSPA1L), in innte immunity (C2, BF, nd C4B), nd in ntigen processing (TAP1, TAP2, nd LMP7). Given the strength of ssocition seen in our dt for the DRB1 locus nd the proposed epitope being consistent with the disese model t this locus, we believe tht DRB1 is most likely importnt s susceptibility locus for JIA. We chose to focus this study on the erly onset groups of ptients. However, the ssocitions of lte onset ptients, both pucirticulr nd polyrticulr, did show very wek ssocitions tht were similr in nture to those found in erly onset ptients. Owing to the limited number of ptients, we were not ble to identify prticulr lleles tht were sttisticlly over-represented in this group. Pending the expnsion of this ptient group, we would propose tht this group of ptients shres some of the susceptibility loci with the erly onset ptients but differs in the complement of other modifying susceptibility loci. The dt from the TNFc locus demonstrte n interesting cution for using the TDT. Normlly, cse control ssocition studies re considered to be fr more powerful thn studies using TDT within nucler fmilies becuse of the number of probnds nd controls tht cn be clerly genotyped in such study. However, ssocition studies re subject to flse-positive results becuse of indvertent strtifiction in either the control or probnd pools. The dt of our TNFc locus my chnge this considertion for some situtions. If the ssocited llele is very frequent in the popultion (prticulrly for billelic mrker) the overll llele frequencies my not be significntly different, but the disequilibrium in trnsmission cn be significnt. Trnsmission disequilibrium (ie the bised trnsmission of one llele over the other to ffected probnds) my then reflect only 1 2% difference in the frequency of lleles between control nd ptient popultions overll, but they my comprise up to 20% of trnsmissions from heterozygous prents to ffected probnds. In this cse, the TDT is more powerful test. This sitution my be present if the true disese locus in question hs smll genotypic risk rtio or is wekly linked to the true disese locus (s we believe it is here). Interestingly, we found in this study strong difference between erly onset pucirticulr nd polyrticulr ptients. For pucirticulr disese, only DRB1, D6S2447 nd D6S2446 nd hplotypes including these loci showed significnt ssocition. There were no significnt cse control hplotype ssocitions (Po0.001) even with close mrkers such s MID108, tht is 75 kb centromeric to D6S2447 (see Tble 1). The polyrticulr group of ptients showed strong ssocition with much more extensive region tht spns MID104/MID108 nd extends 800 kb telomeric to D6S273. Moreover, two locus hplotypes with significnt ssocition (Po0.001) for the polyrticulr probnds extend proximlly over 900 kb to the TNFc microstellite nd centromeric for the MID mrkers s fr s we genotyped t the D6S1616 microstellite, distnce of over kb (10 cm) (Figure 1). A recent study hs found dditionl evidence tht persistent nd extended pucirticulr diseses (or oligorthritis s defined by the Interntionl Legue of Associtions for Rheumtology) re immunologiclly distinct subtypes s well. We did not seprte these groups in this study. Their distinction my hve helped to strengthen ssocition in pucirticulr onset ptients nd to clrify the differences between pucirticulr nd polyrticulr courses of disese. 27 In n ttempt to ssess the effect of the MHC in ptients who did not possess the DRB1 JIA epitope, we strtified our dt by removing pucirticulr fmilies with DRB1*08-positive probnds nd polyrticulr fmilies with DRB1*08 nd DRB1*11 probnds. While the number of probnds becme smll in this strtifiction (19 pucirticulr nd 27 polyrticulr probnds), the nlysis indicted tht the clss two region ws still importnt in these polyrticulr ptients (P ¼ for DRB1, P ¼ for D6S2446, nd P ¼ for D6S2447). DRB1*01 ws the over-represented llele subclss in these probnds nd showed evidence for bised trnsmission in the TDT (P ¼ ). We believe tht the ssocition of DRB1*01 in these ptients my be becuse of LD with other polyrticulr susceptibility loci in this region. The most likely cndidtes would be the HLA-DPB1 or HLA-DQA1 genes. Both of these loci hve shown ssocition with JIA subtypes (see review in Glss nd Ginnini 3 ) but for only DQA1 hs disese-specific

8 muttion been proposed s possible etiology for JIA. 18,28 As n extension of these studies, we re currently typing HLA-DPB1 nd number of dditionl closely linked mrkers. In the MHC region, second locus for susceptibility is suggested by the sttisticlly significnt, lthough less strong, ssocition of the D6S464 microstellite (the 208 bp llele in prticulr) with polyrticulr disese. However, this locus is not ssocited s prt of hplotype with the DRB1 gene or ny of the mrkers surrounding the DRB1 locus tht showed positive ssocition with polyrticulr disese. This my indicte tht the D6S464 locus is linked to susceptibility locus tht is independent of the DRB1 susceptibility, but it my lso result from lck of sttisticl power to resolve hplotype ssocitions with these very highly polymorphic loci. The ssocition of the D6S464 locus my be becuse of linkge with the HLA-A2 gene tht hs been suggested s susceptibility locus in previous studies. 29,30 However, the D6S464 locus is over 5 Mb telomeric of HLA-A2. Since no closer microstellites showed ssocition in this study, ssocition of D6S464 more likely indictes the presence of n dditionl JIA susceptibility locus. We did not type the HLA-A locus nd cnnot demonstrte independence from tht locus here, but similr evidence for dditionl susceptibility loci close to HLA-A2 hs been found recently using the D6S265 microstellite in Norwegin cohort of JIA ptients. 31 The difference in ssocition between the pucirticulr nd polyrticulr subtypes of JIA ws not becuse of differences in sttisticl power. Therefore, this difference my be becuse of one of three possibilities: (1) the polyrticulr group of JIA ptients contins more susceptibility loci in this region of the chromosome thn do pucirticulr probnds; (2) the pucirticulr nd polyrticulr JIA subgroups hve different MHC vrint, tht is disese susceptibility fctor; or (3) the two subtypes re the result of seprte founder events nd linkge between shred susceptibility loci hs decyed differently in the two subtypes. The second possibility, while not excluded, is unlikely since DRB1*08 is ssocited with both subtypes of disese nd the locl hplotype (including D6S2446 nd D6S2447) ssocited is lso the sme. Similrly, the third possibility ppers unlikely since it would require tht the originl founding events not only occurred seprtely for pucirticulr nd polyrticulr disese but tht they hve remined seprted. The grndprents of the JIA probnds in this study cnnot be seprted geogrphiclly bsed on probnd subtype (dt not shown). Given the popultion history of settling in Finlnd, it is unlikely tht such division hs ever existed nd cnnot hve been mintined in the probnds of this study, lthough this would require more detiled genelogicl nlysis. We therefore fvor the first explntion tht dditionl susceptibility loci in the extended MHC re required to generte the polyrticulr subtype of disese. According to our hypothesis, the polyrticulr probnds would hve second gene in LD with DRB1 in the Finnish popultion tht is not necessry for pucirticulr disese. Additionl studies in n expnded cohort of Finnish JIA ptients re currently ddressing other potentil susceptibility genes within nd outside the MHC. Mterils nd methods Ptient nd control smples DNA smples from Finnish probnd trios nd controls were obtined nd prepred from buccl cell swbs or extrcted from peripherl blood s previously described. 32 All smples were quntified in microtiter trys using PicoGreen fluorescence ssy (Moleculr Probes). Smples of JIA ptients were collected t the Rheumtism Foundtion Hospitl in Finlnd. The Rheumtism Foundtion Hospitl receives ptients from ll the five university hospitls in Finlnd s well s from the remining 16 centrl hospitl districts nd lso directly from primry cre clinics in the entire country, but prticulrly in the middle-estern region where the hospitl is locted. It is estimted tht bout two-thirds of ll Finnish JIA ptients re seen t the Rheumtism Foundtion Hospitl t some time. Only those constntly monitored by the Rheumtism Foundtion Hospitl were selectively included in this study series. This group consisted of 235 ptient smples (233 probnds nd one concordnt sib pir) nd 393 prent smples (184 fthers nd 209 mothers) s well s smples from 57 siblings. Of the probnds, 57 were mle nd 178 were femle nd 150 hd onset of disese symptoms before the ge of 6 yers. The 82 pucirticulr onset nd 153 polyrticulr RFnegtive onset probnds were clssified ccording to the criteri of Brewer et l. 33 Probnds ll tested negtive for RF nd HLA-B27-positive individuls were excluded from nlysis. Finnish control individuls were ll 65+ yers of ge, rndomly selected from the popultion nd with no history of JIA or RA. The ethics committee of the Rheumtism Foundtion Hospitl pproved this study nd the ptients/prents gve informed consent to prticipte in the study. Mrker selection Mrkers were selected tht would spn n pproximtely 20 cm section of chromosome 6 tht included ll MHC clss I nd clss II loci. An ttempt ws mde to mximize the potentil informtion content vilble in ech mrker by considering its previous use in relted studies, its mximum heterozygosity score, nd tested ese of mplifiction nd scoring. A totl of 14 microstellites (D6S461, D6S299, D6S464, D6S478, D6S1558, D6S306, TNFc, D6S273, D6S2446, D6S2447, D6S497, D6S291, D6S1641, nd D6S1616) nd two short insertion/deletion polymorphism (SIDP) (MID108 nd MID104) mrkers were selected. Primer sequences for the DS microstellites re s indicted in the Genome Dtbse ( nd tht for the MID mrkers were s described by Mrshfield Genetics ( Primer sequences for the D6S2447 pir of mrkers were synthesized s described in Lin et l. 34 PCR PCR mplifiction for ll mrkers ws performed in 384- well pltes for 30 cycles using 0.5 ml PCR buffer, 0.7 ml 2.5 mm dntp mix (Phrmci & Upjohn), 0.05 ml cdna polymerse (Clonetech Advntge), 0.1 ml 10mM flourescently lbeled primer mix, 2.65 ml double distilled H 2 O, nd 1 ml 0.2 mg/ul genomic DNA for 5 ml totl rection volume. DS microstellites were mplified ccording to 333

9 334 optiml conditions defined by Reserch Genetics. Rections were plced in PE 9700 therml cycler for 2 min t 951C; 30 cycles of 45 s t 941C, 45 s t 571C, nd 1 min t 721C; nd finl extension of 7 min t 721C. TNFc mplifiction conditions (modified from Udlov 35 ) were 3 min t 951C; 30 cycles of 30 s t 941C, 1 min t 601C, nd 1 min t 721C; nd finl extension of 10 min t 721C. MID mrkers were mplified with optiml PCR conditions described by Mrshfield ( mrshfieldclinic.org/genetics/). D6S2447 mrkers were mplified ccording to Lin et l. 34 Genotyping PCR mplified product (B0.5 ml) of ech mrker ws run on the ABI 3700 DNA Anlyzer. Genotyping ws performed using nlysis softwre provided (GeneScn nd Genotyper from Perkin Elmer/ABI). DRB1 typing The DRB1 locus ws typed using AlleleSEQR HLA- DRB1 Sequencing-Bsed typing kit (Forensic Anlyticl, Hywrd, CA 94545, USA) ccording to the mnufcturer s instructions for the ABI 3700 DNA Anlyzer. This method directly sequences exon 2. Sequence nlysis ws performed using the supplied ABI softwre (SeqEd, Mtchtools, nd MT Nvigtor). Anlysis For ech locus, w 2 nlysis for ssocition by llele nd by genotype ws performed using the softwre CLUMP 36 ginst n identiclly typed popultion of Finnish control individuls. We used CLUMP to evlute 2 m 0 contingency tble in which n originl tble with m columns (equivlent to the number of lleles) ws trnsformed into new tble with m 0 columns by combining columns with smll vlues. If ny cell hd n expected vlue less thn 5, then its vlue for both control nd ptient ws lumped together with the cells in the column with the next smllest totl until every cell hs n expected vlue of 5 or more. The significnce of the w 2 vlue obtined from this tble in the CLUMP lgorithm is evluted using the Monte Crlo method nd should closely correspond to the nominl significnce of the w 2 sttistic with m 0 1 degrees of freedom (where m 0 is the number of columns in the clumped tble). For the Monte Crlo simultions, tbles with 2-bym 0 cells re simulted with row nd column totls constrined to be the sme s for the clumped tble (not the originl 2- by-m tble). The number of times simulted tble yields w 2 vlue greter thn or equl to tht obtined from the clumped tble is output. Odds rtios (ORs) were clculted by the Mntel Henszel odds rtio nd 95% confidence intervl compiled on the internet ( epitools/rfunctions/ormh.html). Anlysis by TDT ws performed using FBAT. 37,38 DRB1 lleles found to be significntly decresed by w 2 (dt not shown) were djusted for compenstory decrese becuse of overrepresented lleles nd renlyzed by w Hplotype nlysis ws performed using the EM lgorithm contined in the EH+ softwre. 39,40 Acknowledgements Dr Runstdler is supported by n NIH postdoctorl fellowship s prt of the trining grnt in Comprtive Medicine t the University of Cliforni, Dvis. This work ws supported in prt by NIH Grnt no. 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