Annls of the Rheumtic Diseses 1994; 53: 587-592 587 Deprtment of Orthopedic Surgery, Knsi Medicl University, Otokoym Hospitl, Kyoto, Jpn Y Tod Y Mori Deprtment of Orthopedic Surgery, Knsi Medicl University, Osk, Jpn Y Minmikw S Akgi H Sugno H Nishimur R Ogw Deprtment of Medicl Sttistics, Knsi Medicl University, Osk, Jpn S Arit Lbortory of Moleculr Biology, Knsi Medicl University, Osk, Jpn Y Sugino Correspondence to: Dr Tod, Deprtment of Orthopedic Surgery, Knsi Medicl University Otokoym Hospitl, zumi 19 Otokoym, Ywt-shi, Kyoto, 614, Jpn. Accepted for publiction 24 My 1994 Rheumtoid-susceptible lleles of HLA-DRB 1 re geneticlly recessive to non-susceptible lleles in the progression of bone destruction in the wrists nd fingers of ptients with RA Yoshitk Tod, Yoshitk Minmikw, Shigeo Akgi, Hiroshi Sugno, Yoshiki Mori, Hideki Nishimur, Seizburo Arit, Yoshinobu Sugino, Ryokei Ogw Abstrct Objective-To ssess the reltionship between HLA-DRB1 genotypes nd the progression of bone destruction in Jpnese ptients with RA. Methods-The HLA-DRB1 lleles were determined by polymerse chin rection nd llele specific oligonucleotide probe techniques in 16 Jpnese ptients with RA. HLA-DR 11, 41, 44, 45, 11 nd 142 were regrded s susceptible lleles of RA ccording to previous reports. Ptients were clssified into three groups (S/S, S/N nd N/N group), bsed on the possession of two, one or no susceptible fctor. The grding of rdiogrphic chnges in the wrists nd fingers were evluted by Lrsen's criteri. The rdiogrphic grdes were first compred with the results of genotyping in the 16 cross sectionl cses. A retrospective study ws then conducted on subgroup consisting of 57 cses tken from the 16 cses used for the cross sectionl study. Results-n the sctter digrm of the 16 cross sectionl cses expressing the reltionship between the stge of bone destruction nd durtion of RA, the regression line nd the 95% confidence intervls seprted the S/S group from the S/N nd N/N groups in the erly phse of development of bone destruction. n the retrospective study on the 57 cses the medin yers tken to development to stge V in the wrists fter the onset of symptoms were 13-1 in the ptients in the S/S group, 22-7 in the S/N group nd 23- in the N/N group. The difference observed between the S/S nd S/N group, nd between the S/S nd N/N group were sttisticlly significnt (p < -1), but tht between the S/N nd N/N groups ws not. Thus the bone destruction in the wrists nd fingers progressed more rpidly in the S/S group thn in the S/N nd N/N groups; nd the rheumtoid susceptible lleles of HLA- DRB1 cn be considered to be geneticlly recessive to the non-susceptible lleles in the progression ofbone destructions in the wrists nd fingers. Conclusion-Genotyping of HLA-DRB1 cn be useful prognostic mrker in the erly phse of RA. (Ann Rheum Dis 1994; 53: 587-592) The clinicl courses of bone destruction in rheumtoid rthritis (RA) vry widely. Even when the ptients re treted in similr wys, in some cses the bone destruction my progress slowly to the end stge in the course of severl decdes, wheres in others it my rpidly dvnce to bone nkylosis or resorption chnge in few yers. t seems very likely tht genetic fctors re involved in the development of bone destruction in RA. Recently, Weynd et l reported tht ptients with RA with two disese linked sequences (tht is, susceptible fctors) in both HLA- DRB 1 lleles were ffected more severely thn other groups in terms of the frequency of nodulr disese, mjor orgn filure nd requirement of joint surgery.3 However, they did not discuss the reletionship between HLA-DRB 1 genotypes nd rpidity with which the disese progresses. t hs been reported tht in most ptients with RA the bone destruction of wrists nd fingers progresses within decde fter onset of RA.4 We were interested in the gents tht ffect the rpidity of the bone destruction in the erly phse of RA. We proposed tht if genotypes of the HLA-DRB 1 lleles re ssocited with the clinicl courses of bone destruction in RA, then genotyping the HLA- DRB1 lleles during the erly phse of the disese might llow prediction of the progression of bone destruction. n this study HLA-DRB1 llele genotyping ws performed in Jpnese ptients with RA nd the results were compred with the dt for helthy Jpnese subjects. The ssocition of the number of susceptible fctors with the clinicl course of bone destruction in the wrists nd fingers ws lso studied. Only rdiogrphic chnges in the wrists nd fingers were ssessed, becuse the bone destruction chnges in these sites is common nd typicl feture of RA, so tht pst records were more frequently vilble. Mterils nd methods One hundred nd sixty Jpnese outptients nd hospitl ptients in the Knsi Medicl University Hospitl with rheumtoid fctor level over 2 U/dl t present or pst nlysis (tht is, seropositive RA), nd dignosed with RA bsed on the 1987 ACR criteri were enrolled in the study.5 For ll ptients the reltionship between the present stte of
588 Tod, Minmikw, Akgi, et l rdiogrphic bone destruction of the wrists nd fingers nd the durtion fter the onset of symptoms ws ssessed. Of the 16 ptients, 57 hd rdiogrphic records of progressive chnge in bone destruction ofthe wrists nd fingers for more thn five yers nd they were reviewed for retrospective study. The genomic DNA ws isolted from mononucler cells in peripherl blood ccording to the method of Erlich nd Bugwn; HLA-DRB1 DNA ws mplified with the primer sets by polymerse chin rection (PCR).6 Ech HLA-DRB1 llele ws genotyped by reverse dot blotting by hybridistion with sequence-specific oligonucleotide. f the HLA-DRB1 lleles were from ptients who ppered to be homozygous, justifiction of homozygosity ws evluted by HLA-DQA1 nd DQB1 genotyping.7 The genotyping of HLA-DRB 1 lleles ws undertken by Shionogi Biomedicl Compny. DSTRBUTON OF HLA-DRB1 The gene frequencies of H[A-DRB 1 lleles in our ptients with RA were compred with the dt for helthy Jpnese subjects.8 According to the previous reports on the ssocition between HLA-DR lleles nd RA, HLA-DRB1 41, 44, 45, 11, 11 nd 142 were regrded s susceptible lleles of RA in the study.' 2 The ptients were 11- iv- 1 The S/S groujp ---..'The S/N group - lhe N. N group A F A C Firigers) VVrrsts 15 2 3 4 5 clssified into three groups. The S/S group consisted of those with susceptible fctors in both of the HLA-DRB1 lleles. The S/N group were mde up of those hving one susceptible fctor nd one non-susceptible fctor. The N/N group consisted of those possessing two non-susceptible fctors. RADOGRAPHC CHANGE Rdiogrphs were ssessed by five orthopedic surgeons before they were informed of the results of genotyping. The stge of bone destruction in the wrist nd finger joints ws ctegorised into six rdiogrphic groups ccording to the stndrd films of Lrsen.9 The stging of fingers ws bsed on the joint which ws ffected most severely. RELATONSHP BETWEEN BONE DESTRUCTON AND TYPES OF HLA-DRBl n the cross sectionl study on the 16 cses, the reltionship between the durtion of RA nd the stge of bone destruction in the wrists nd fingers in the S/S, S/N nd N/N groups is illustrted on the sctter digrms (fig 1). Regression lines nd their 95% confidence intervls were lso represented in the sctter digrms of the three groups, tking the stge numbers s numericl vlues. As we cn not be certin tht the intervls between stges should be equl in truly quntittive wy, we dded V V- 111- B (Fingers) o 4_.C.7' 7 77 BZ ' '-7A n] 1 2 Ml5 X D o,/ (Wrists) AA7; ZXZ! 1 2 3 4 5 1 2 3 4 5 Yers fter onset of symptoms Figure 1 Sctter digrms showing the reltionship between the durtion ofra nd the stge of bone destruction in the wrists ndfingers, with the regression lines nd 95% confidence intervls. A) The S/S nd S/N groups of thefingers; B) The S/S nd N/N groups of the fingers; C) The S/S nd S/N groups of the wrists; D) The S/S nd N/Ngroups of the wrists. Ech dot represents n individul, but if the dots of the sme group overlp, only one dot is represented on the digrm.
Rheumtoid-susceptible lleles of HLA-DRB 589 Tble 1 Ptient chrcteristics of the 16 cses exmined, nd the 57 reviewed cses 16 cses exmined 57 reviewed cses All S/S group S/N group N/N group All S/Sgroup S/Ngroup N/Ngroup (n= 16) (n=32) (n= 78) (n=5) (n=57) (n=1) (n=27) (n=2) 1 Sex 1 Sex Femle 139 3 64 45 Femle 5 9 23 17 Mle 21 2 14 5 Mle 7 1 4 3 2 Age 2 Age <39 12 2 7 3 <39 3 1 1 1 4-49 28 4 17 7 4-49 9 1 6 2 5-59 6 16 28 16 5-59 22 6 9 7 6-69 32 5 12 15 6-69 12 1 6 5 7-79 24 4 12 8 7-79 1 1 4 5 >8 4 1 2 1 >8 1 1 medin 56 55 54 58 medin 56 55 57 59 3 Disese 3 Disese durtion durtion -4 43 8 25 1 5-9 44 12 17 15 5-9 21 4 1 7 1-14 23 2 1 11 1-14 1 1 4 5 15-19 18 3 1 5 15-19 1 2 4 4 2-24 18 2 1 6 2-24 1 2 5 3 <25 14 5 6 3 <25 6 1 4 1 medin 8-5 8-8- 9 5 medin 13-12-5 14-11-5 the ssessment of the contingency tbles showing the reltionship between the stges of bone destruction nd the different groups s sttisticl support dt. n the retrospective study on the 57 reviewed cses, the chnges in stge of bone destruction with yers were studied nd compred mong the S/S, S/N nd N/N groups. Reversion of stge of bone destruction ws not observed; for exmple, stge never recovered to stge or. The end stge ws stge V which is nlogous to 'deth' on the sttisticl life tble. We showed the 'life tble' of development to stge V. STATSTCAL ANALYSS The Chi-squre test ws used to determine the sttisticl significnce of the differences in the sex, ge nd disese durtion between the 16 Tble 2 Distribution ofgenes in both HLA-DRB1 lleles in RA The S/S group (n = 32) 142 11 45 44 41 11 11 8 1 41 1 1 1 44 45 2 18 11 142 The S/N group (n = 78) 42/-3/-6/-7 82/3 91 111/2 121/2 131/2 141/3/5151/2 162 /-9/-1 11 3 4 5 5 41 1 1 1 1 1 3 44 45 3 1 11 2 3 1 8 21 1 11 1 142 1 The N/N group (n = 5) 162 151/2 141/3/5 131/2 121/2 111/2 91 82/3 42/-3/-6/ -7/-9/-1 42/-3/-6/ 3 1 1 1 2-7/-9/-1 82/3 2 4 2 1 5 91 1 2 2 3 1 9 111/2 2 121/2 1 131/2 141/3/5 1 2 151/2 4 162 cses exmined in the cross sectionl survey on the one hnd, nd the 57 cses selected for the retrospective study on the other; the differences between the RA nd helthy subjects in gene frequencies of HLA-DRB1 lleles; nd the differences in stge of bone destruction mong the S/S, S/N nd N/N groups in the contingency tbles. The Kpln-Meier method ws used to ssess the 'survivl' probbility tht the bone destruction did not progress to stge V,' nd the significnce of the differences in the probbility mong the three groups ws ssessed by the log-rnk test of Peto.' Results POPULATONS OF SUBJECTS One hundred nd sixty ptients, 139 women nd 21 men, rnging from 23 to 86 yers of ge (medin, 56), were studied for n initil cross sectionl study. The durtion of disese rnged from -5 to 46 yers (medin, 8 5). The 16 cses were composed of 32 cses in the S/S group, 78 cses in the S/N group nd 5 cses in the N/N group (tble 2). Of the 16 cses, 57 cses for which pst rdiogrphic records for more thn five yers were vilble, were selected for further retrospective study. This group of 57 reviewed cses were not significntly different in the medins nd rnges of ge nd disese durtion from the initil group of 16 cses (p < 5), nd consisted of 1 cses in the S/S group, 27 cses in the S/N group nd 2 cses in the N/N group (tble 1). DSTRBUTON OF HLA-DRB1 ALLELES The gene frequencies of the HA-DRB 1 lleles in ptients with RA nd helthy Jpnese subjects re shown in tble 3. The susceptible fctor which ws found in high frequency in ptients with RA in this study ws HLA-DRB 1 45, lthough it is lso comprtively common llele in helthy Jpnese subjects. The frequency of 45 ws very much higher in our ptients with RA thn in helthy Jpnese subjects (n = 493), (p < - 1). The frequencies of the other
59 Tod, Minmikw, Akgi, et l Tble 3 The gene frequencies ofhla-drbl lleles in the ptients with RA nd helthy Jpnese subjects Susceptible lleles 11 41 44 45 11 142 RAptients (n= 16) 8-8% 3-1% % 31-3% -6% -6% Helthy Jpnese subjects (n = 493) 5% 1-8% -7% 12-5%.9%.5% Non-susceptible lleles 42/3/6 82/3 91 111/2 121/2 131/2 141/3/5 151/2 162 Others /7/9/1 RAptients (n= 16) 4-7% 6-9% 16-3% 1-9% 3-7% 1-9% 5-4% 14-1% -9% % HelthyJpnese subjects (n = 493) 7-5% 11-5% 13-7% 2-2% 5.9% 4.9% 6-9% 16-% 77% 9-3% susceptible fctors (HLA-DRB 1 11,41, 44, 11 nd 142) in the RA ptients in this study were seprtely not high enough to llow significnt comprison with helthy Jpnese subjects, but when they were dded together, the sum ws significntly higher thn the corresponding sum for helthy Jpnese subjects (p < 5). RELATONSHP BETWEEN BONE DESTRUCTON AND TYPES OF HLA-DRB1 A The cross sectionl study n the sctter digrms showing the reltionship between the stge of bone destruction in Tble 4 The life tbles indicting the observtion period nd development to stge V in the 57 reviewed cses The S/S group (n = 1) The S/N group (n = 27) * Stge V in the wrist * Stge V in the fingers L - Rdiogrphic reccording yers l l~~~~~~ tt e ALJ LL. lj l~ 1 2 3 s T Yers fter onset of symptoms m the wrists nd the durtion of RA, the regression lines nd their 95% confidence intervls of the three groups re represented in (fig 1). Both in the wrists nd fingers, the 95% confidence intervl of the S/S group did not overlp with tht of the S/N group in the erly phse when bone destruction progressed most rpidly (fig 1-A, C). The 95% confidence intervls of the S/S nd N/N groups lso did not overlp in the erly phse of progression of bone destruction (fig 1-B, D). However, the 95% confidence intervls of the S/N nd N/N groups overlpped for the whole rnge of yers fter RA onset in both the fingers nd the wrists. As mentioned in the ntroduction, bone destruction of wrists nd fingers in RA progresses most rpidly within the first decde of the erly phse. We therefore constructed the contingency tbles of the cses with RA for less thn decde (dt not shown). Significnt differences in distribution of the stges were observed between the S/S nd the S/N groups (p = -316 in the wrists, -86 in the fingers) nd the S/S nd N/N group (p = -226 in the wrists, -15 in the fingers), but not between the S/N nd N/N group (p =-627 in the wrists, -189 in the fingers). B The retrospective study The life tble of development to stge V of the 57 reviewed cses is illustrted (tble 4). The observtion periods were longer thn five yers, beginning t vrious times fter onset of the disese, nd t lest three rdiogrphs tken t different time points were ssessed in ll cses. The progression of bone destruction in the fingers in the S/S group ws more rpid thn in the S/N nd N/N groups s shown by the survivl probbility tht the bone destruction did not progress to stge V (fig 2A). n this grph ll cses of the S/S group progressed to stge V within 17 yers fter RA onset. The medin yers tken to develop to stge V fter the onset of symptoms of the fingers determined by the log-rnk test were 13d1 for the S/S group, 22-7 for the S/N group nd 23- for the N/N group. The differences were sttisticlly significnt between the S/S nd S/N groups nd between the S/S nd N/N groups (both p < 1), but not between the S/N nd N/N groups (p > -5). Similr sttisticl differences between the respective groups were observed in the bone destruction of the wrists (fig 2B). Thus the
Rheumtoid-susceptible lleles of HLA-DRB591 11). ) 4-. -. C', -co H3 >z g ) Q) cn o 2 v ux O O (%) 2 C * 4- en CD) - 1 2 Yers fter onset of symptoms Figure 2 The 'survivl' probbility tht the bone destruction did not progress to stge V bsed on tble 4. A) n the fingers; B) n the wrists. medin yers tken to development to stge V in the wrists were 13-1 in the S/S group, 23-1 in the S/N group nd 23 in the N/N group. Discussion Severl studies using immunologicl nd serologicl techniques hve reveled tht HLA- DR4 ntigen is ssocited with more progressive rdiogrphic bone destruction in ptients with RA.'2-13 Recently, genotyping by PCR nd oligonucleotide probe techniques hs been pplied for ssessing this ssocition.314 Emery reported tht erly symmetricl (rheumtoid-like) rthritis ptients with the conserved clss mjor HLA complex genes ssocited with RA (susceptible fctors) were t reltively high risk for the development of rdiogrphic erosions prospectively. "4 n our cross sectionl nd retrospective studies clssifiction by the number of susceptible fctors present in the lleles ws used to evlute the ssocition with progression of bone destruction in ptients with RA. Two conclusions emerge from this study, which re closely relted; one is tht the results of genotyping using this clssifiction cn be useful prognostic mrker in the erly phse of RA, nd the other is tht rheumtoid susceptible lleles of HLA-DRB 1 re geneticlly recessive to non-susceptible lleles with respect to the progression of bone destruction in the wrists nd fingers. A HLA-DRB1 lleles s prognostic mrker n our cross sectionl study the difference between the S/S group nd the S/N group ws significnt, s ws tht between the S/S group nd the N/N group, but the difference between the S/N group nd the N/N group ws not significnt, in the erly phse of development of bone destruction. n the retrospective study the medin yers tken to development to stge V fter the onset of symptoms were significntly smller in the ptients in the S/S group thn in the S/N group nd in the N/N group. The conclusion from these dt is tht, when the erly phse of seropositive RA is dignosed in ptient who hs two susceptible fctors in both HLA-DRB 1 lleles, the investigtor cn predict tht bone destruction in the wrists nd fingers will dvnce within reltively shorter period fter the onset of symptoms, thn in ptient who hs only one or no susceptible fctor. He or she should pln the clinicl mngement corresponding to the nticipted course. The role of rheumtoid fctor s prognostic fctor hs been exmined previously. 1`-6 Bums nd Clin reported tht rdiogrphic bone destruction in the wrists nd hnds of the seropositive group differed significntly from those of the seronegtive group."6 n this study only seropositive cses were selected to ssess the bone destruction chnge. We re currently trying to ssess the reltionship between bone destruction nd HLA-DRB1 lleles in seronegtive cses. But the bone destruction of seronegtive cses is not so esy to ssess s in seropositive cses, s Bums nd Clin hve reported. Lrsen et l9 lso reported tht his criteri ws very useful for seropositive rheumtoid rthritis, but not for seronegtive rthritis. The merits of genotyping s mens of predicting dignosis re tht the result remins unchnged throughout life nd tht it cn be determined in the erly phse of the disese. We believe tht the genotyping test holds some promise s prognostic mrker in the erly phse of RA. B Recessiveness ofsusceptible lleles The reltionship between HA-DRB1 lleles nd the etiology of RA is still uncler, lthough severl hypotheses hve been proposed.'7-18 t is relevnt whether the susceptible lleles re dominnt over or recessive to the non-susceptible lleles. Wynd et l noted tht ptients with HLA-DR4 homozygosity hve higher risk for more severe disese thn the ptients with heterozygosity for susceptible nd non-susceptible lleles. However, they did not discuss the dominnce or recessiveness of susceptible lleles.3 Our dt clerly indicted tht rheumtoid susceptible lleles of HLA- DRB 1 re geneticlly recessive to nonsusceptible lleles with respect to the progression of bone destruction in the wrists nd fingers. n ptients ffected by RA for less thn decde, the distribution of the stges were significntly different between the S/S nd the S/N group, nd lso between the S/S nd N/N group, but not between the S/N nd N/N group, both for the wrists nd fingers. The medin yers tken to develop to stge V in the wrists nd fingers were shorter in the S/S group thn in the S/N nd N/N group, wheres significnt differences were not detected between the S/N md N/N group.
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