HLA-DR Genotype Risks in Seropositive Rheumatoid Arthritis

Am J Hum Genet 36:690-699, 1984 HLA-DR Genotype Risks in Seropositive Rheumatoid Arthritis L. LEGRAND,1 G. M. LATHROP,2 A. MARCELLI-BARGE,' A. DRYLL,3 T. BARDIN,3 N. DEBEYRE,3 J. C. POIRIER,' M. SCHMID,' A. RYCKEWAERT,3 AND J. DAUSSET1 SUMMARY We studied the distribution of HLA-A, B, C, and -DR antigens in 77 Caucasian patients with sero-positive rheumatoid arthritis. Forty-four patients were genotyped and compared with the control panel of 110 unrelated Caucasian genotyped donors. The data obtained confirm the association of DR4 with RA, and reveal an increased risk of disease for patients carrying DRI, DR2, and DR3, compared to the risk for those carrying other antigens, such as DR5, DRw6, and DR7. There is a higher risk for DR414 homozygotes than for DR411, DR412, or DR413 heterozygotes. DR415, DR416, and DR417 have a lower risk than the previously mentioned genotypes. The genotype risks are compatible with the inheritance of a single, linked genetic determinant of disease susceptibility, but we are unable to distinguish between recessive and dominant inheritance of susceptibility using the "antigen-frequenciesamongst-diseases" method. DR4 seems to be more frequent in patients in whom onset occurs before the age of 35 (79% vs. 54% DR4 positive). A significant excess of DR3 + is observed in patients with toxic complications following treatment with gold salts (X21 = 8.96). INTRODUCTION A positive association between HLA-Dw4 and rheumatoid arthritis (RA) in Caucasian patients was first reported by Stastny [1]. This finding has subsequently been confirmed in other studies [2, 3]. In previous studies [4, 5], including those of the 7th and 8th International Histocompatibility Workshops [6], HLA-Dw4 Received August 11, 1983; revised December 5, 1983. INSERM U.93, H6pital St. Louis, 2 place du Dr Fournier, 75475 Paris, Cedex 10, France. 2 INSERM and Laboratoire d'anthropologie, Musee de l'homme, Paris, France. 3 Clinique de Rhumatologie, H6pital Lariboisiere, 6 rue Guy Patin, 75010 Paris, France. C 1984 by the American Society of Human Genetics. All rights reserved. 0002-9297/84/3603-0019$02.00 690

HLA-DR GENOTYPE IN RHEUMATOID ARTHRITIS and DR4 were found to be increased in adult Caucasians with erosive seropositive RA; whereas in seronegative RA patients, there was no excess of this antigen. Weaker positive or negative associations with other DR antigens have also been reported in some studies [2, 7]. Surveys of unrelated adult RA patients in other populations have shown a highly significant increase of HLA-DR4 except in Jewish and Asian Indian groups [7, 8]. The increased risk associated with DR4 may be due to linkage disequilibrium with a susceptibility gene(s) within the major histocompatibility complex. Family studies [7, 9, 10] have demonstrated a strong association of familial RA with DR4, and suggest that, at least in some families, HLA-associated genetic susceptibility factors are contributed by both affected and unaffected parents. The purpose of this study was to evaluate the frequency of HLA-A, -B, -C, and -DR antigens and genotypes in a series of 77 patients with seropositive adultonset RA. This study is the first to investigate the risks associated with particular genotypes. Patients MATERIALS AND METHODS Seventy-seven unrelated Caucasian patients (61 female and 16 male), aged 29-93 years, with classical or definite rheumatoid arthritis according to the ARA criteria [11] were studied. Detailed clinical information was collected on all patients included in the study. All patients had positive titres for the rheumatoid factor (> 1:80 by the latex fixation test). In 24 cases, the disease was complicated by Sjogren syndrome. Sixty-seven patients were treated with sodium aurothiopropanol sulfonate, and 29 also received penicillamine. All cases were reviewed to evaluate tolerance to this treatment. Toxic reactions were monitored by periodic urine analysis, blood counts, and physical examination. Direct questioning was used to elicit a family history of RA and the age of onset of the disease. HLA Typing HLA-A, -B, and -C antigens were identified with the conventional microcytotoxicity test in which well-validated antisera were used. For HLA-DR antigen determinations, enriched suspensions of B-cells were prepared as described [12]. The control panel of white-cell donors consisted of 110 genotyped, unrelated Caucasians with a similar age and sex distribution. Patients and controls were all of French origin, living in the Paris region. In addition, HLA typing was performed on the families of 44 patients. HLA genotypes were determined for these individuals. Statistical Analysis 691 Association has been investigated with the "antigen-frequencies-amongst-diseases" method [13], extended to multiple antigens, and allowance made for a "blank" allele. Susceptibility is assumed to be determined by a single locus with the alleles D (called the "disease allele") and d, with frequencies p and 1 - p, respectively. The probabilities of disease, or penetrances, for the genotypes DID and Dld are unknown; did is assumed to have no risk of disease. p(aid) is the proportion of D chromosomes also carrying A where A represents a DR antigen. Similarly, p(ald) is the proportion of d chromosomes carrying a DR A antigen. Linkage disequilibrium or association exists if p(aid) # p(ald). When probability (p) is low and mating has been random, genotypes in controls have Hardy- Weinberg proportions with antigen frequencies p(ald). Genotype frequencies in patients are as follows: If A = B: F(AIB) = 2 (1 - k)p(aid)p(bid) + 0.5 k[p(aid)p(bld) +

692 LEGRAND ET AL. P(AId)P(BID)]. Otherwise: F(AIB) = (1 - k)p2(aid) + k P(AID)P(AId), where k is the proportion of Dld genotypes in patients (a function of the unknown penetrances and p ). If D is dominant, k is approximately 1, and if D is recessive k = 0. Cases positive for a single A antigen may have either the genotype DRAIA or DRAlblank. If genotypes have not been determined using family studies, the frequency of this phenotype amongst cases is: f (Alcases) = f (A/blanklcases) + f (A/Alcases). Parameter estimates have been obtained by maximum likelihood, and tests have been done using the likelihood ratio statistics [14]. The null hypothesis for the test of association TABLE 1 HLA-A, -B, and -C GENE FREQUENCIES ESTIMATED IN PATIENTS AND CONTROLS ESTIMATED FREQUENCIES HLA ANTIGENS Patients Controls HLA-Al..18.13 A2..36.28 A3..09.12 All.06.07 Aw23..02.04 Aw24..05.10 Aw25..01.02 Aw26..05.07 A28.06.04 A29.06.04 Aw3O..01.05 Aw3l..05.02 Aw32..03.05 Aw33. 0 0 "Blank". 0.01 HLA-B7..16.12 B8..14.10 B13. 01.02 B 14. 01.05 B 15. 08.05 B 17. 03.03 B 18. 06.08 B21.05.04 B27. 02.05 B35. 06.09 Bw38...06.01 Bw39...01.02 B40.06.05 Bw4l...01.01 Bw44.. 15.15 Bw45.. 0.01 Bw5l...05.06 Bw52...01 Bw53.. 0 0 Bw54...02 0 BwS5...01.03 "Blank"..02.02 HLA-Cwl.04.04 Cw2.05.08 Cw3 1 3.07 Cw4.09.12 Cw5.11.10 Cw6.03.07 Cw7.29.20 "Blank"..26.33

HLA-DR GENOTYPE IN RHEUMATOID ARTHRITIS TABLE 2 HLA-DR GENOTYPES IN 77 PATIENTS WITH RHEUMATOID ARTHRITIS (RA) 693 Single DR] DR2 DR3 DR4 DRS DRw6 DR7 DR8 "Blank" antigen* DR].0 2 1 8 1 0 0 0 DR2.. 0 6 11 2 0 1 0 1 1 DR3.. 0 6 3 1 3 0 3 0 DR4 2 6 2 4 4 6 DR5.- - * 0 0 0 0 DRw6... 0 0 0 DR7..... 0 0 0 1... DR8. Blank... 0 1.*-- 0 0 NOTE: The no. patients observed for each possible pair of antigens is given in the upper triangle. Ungenotyped patients positive for a single antigen are shown in the row for the appropriate antigen and the column labeled "single antigen." * Nongenotyped individuals positive for a single antigen. is P(AID) = P(AId) for all antigens, and the alternative is P(AID) # P(AId) for one or more A. Estimates for the dominant or recessive models are found by fixing k = 1 (dominant hypothesis) or k = 0 (recessive hypothesis). The disease risk of antigen A, relative to antigen B, is: RA,B p (Ajcases) p (Blcontrols) p (Blcases) p (Alcontrols) where p (Alcases) = Ef(A/CI cases) + 2f (A/Alcases) with the sum taken over all C =# A, and p (Alcontrols) = p (Ald). Genotype and phenotype risks are calculated similarly. If RA.B = 1, the relative risk for the genotypes DR AIC and DR BIC, where C is any antigen (denoted RA/C B/C, is also 1. Hence, antigens are grouped to obtain a combined estimate of the genotype risks when the hypothesis RAB = 1 is accepted. RESULTS HLA-DR Antigens and Rheumatoid Arthritis Table 1 shows the gene frequencies of HLA-A, -B and -C in patients and controls. Tables 2 and 3 give the distribution of HLA-DR phenotypes in patients TABLE 3 HLA-DR GENOTYPES IN 1 10 HEALTHY CONTROLS DR] DR2 DR3 DR4 DR5 DRw6 DR7 DR8 "Blank" DR]. DR2. 0 2 0 1 4 2 4 4 7 1 5 5 3 0 1 0 8 DR3 2 0 5 0 4 0 5.. DR4. 0 3 1 2 0 5 DR5 3 2 4 0 7 DRw6. 1 1 0 3 DR7. - DR8. c o v "Blank". r ec.. p b p * - - 10 i t - - I- NOTE: The no. controls observed for each possible pair of antigens is given in the upper triangle.

694 LEGRAND ET AL. TABLE 4 HLA-DR GENE FREQUENCIES ESTIMATED IN PATIENTS AND CONTROLS ESTIMATED FREQUENCIES HLA ANTIGEN Patients Controls DRI..08.07 DR2..15.15 DR3..17.11 DR4...35.08 DR5..08.17 DRw6...03.07 DR7..07.15 DR8. 0.01 "Blank".10.18 and controls, respectively. As observed at the 8th International Workshop [7], a substantial and highly significant increase in DR4 is found in patients. In addition, the estimated antigen frequencies given in table 4 show a decreased frequency of DR5, DRw6, DR7, and DRw8 and "blank" antigens in cases. The frequencies of DRI, DR2, and DR3 are similar in the two groups. The test of association between the DR antigens and RA gave significant results (X28 = 72.63). (DR8 was not considered in this test because this antigen has a low frequency in controls and is present amongst cases.) We are unable to reject either the dominant (k = 1) or recessive (k = 0) hypotheses against the intermediate model (k = 0.89). Thus, despite the strong association with DR4, these data are not informative with regard to the mode of inheritance. The goodness of fit to the observed phenotype frequencies shows no significant departure from the model (X2 24= 20.16). Relative Risks Estimated antigen risks relative to the "blank" antigen are given in table 5. After accounting for association with DR4, there is significant heterogeneity TABLE 5 HLA-DR ANTIGENS AND ESTIMATED RELATIVE RISKS IN RHEUMATOID ARTHRITIS (RA) HLA antigen Estimated risk DRI.2.19 DR2. 1.78 DR3.2.86 DR4.8.47 DR5.0.82 DRw6.0.81 DR7.0.90 NOTE: Risks are given relative to the "blank" antigen and are calculated from the allelic frequency estimates in table 4.

HLA-DR GENOTYPE IN RHEUMATOID ARTHRITIS 695 TABLE 6 ESTIMATED GENOTYPE RISKS RELATIVE TO DRX/X Genotype Risk DR414.. 124.46 DR41Y. 31.89 DR41X. 11.16 DRY/Y. 8.17 DRYIX. 2.86 NOTE: In this table, X refers to the antigens DR5, DRw6, DR7, and "blank," and Y refers to the antigens DRI, DR2, and DR3. amongst the risks for the other alleles (X26 = 29.67). The antigens DR5, DRw6, and DR7 have similar relative risks. The relative risks in this group do not differ significantly from 1 (X22 = 2.78). We will denote DR5, DRw6, DR7, and the "blank" antigen as DRX. The antigens DRI, DR2, and DR3 form a second group intermediate between DR4 and DRX. The risks for DRI, DR2, and DR3 are not statistically heterogeneous (X22 = 0.99), but each differs significantly from 1 (X21 = 9.60 for DRI; X21 = 10.63 for DR2; X2i = 17.38 for DR3). An antigen from this second group will be denoted as DRY. Under the constraint of equal risks within the antigen groups, the estimate of k is 0. The genotype risks relative to DRXIX as calculated from the estimated frequencies are shown in table 6. The estimated phenotype risks (combining homozygotes and heterozygotes carrying the "blank" antigen) are similar to those obtained from the observed phenotype frequencies (table 7). Because there is only one DRXIX patient, this phenotype is excluded from table 7, and the risks are shown relative to DRYIX. The goodness of fit can be determined by comparing the likelihood under the genetic hypothesis with that obtained when unconstrained risks are estimated for each genotype category. Departures from the genetic hypothesis are not significant (X22 = 6.76). TABLE 7 A COMPARISON OF ESTIMATED AND OBSERVED PHENOTYPE RISKS RELATIVE TO DRY/X Phenotype Observed* Estimated DR4 (DR414 and DR41b combined) 8.70 11.92 DR41Y. 11.83 11.15 DR41X (except DR4/b).. 4.69 3.90 DRY (DRYlY and DRYlb combined) 1.59 1.88 NOTE: In this table, DRX stands for DR5, DRw6, DR7, or "blank," and DRY stands for DR], DR2, and DR3. The phenotype DR4 combines the genotypes DR414 and DR41"blank." Similarly, DRY combines DRYlY and DRYI/blank." DRXIX is excluded from the table because only one such genotype was found amongst patients. * Calculatedas(a + 0.5)(d + 0.5)/(C + 0.5)(b + 0.5), whereaisthe frequencyofthegenotype in patients, b is its frequency in controls, c is the frequency of DRYIX (except DRYI/blank") in cases, and d is the frequency of DRYIX (except DRYI/blank") in controls. b = "blank."

696 LEGRAND ET AL. Association between HLA-DR Phenotypes and Clinical Variables Because of reports that DR4 may be associated with particularly severe disease and early onset of RA [3, 6, 15-18], we examined the association between this antigen and clinical or laboratory measures on patients. DR4 is more frequent in patients in whom onset of the disease occurs before age 35 (79% vs. 54% DR4 positive; X21 = 4.36). This antigen is also more common in pure rheumatoid arthritis than in forms associated with Sjogren syndrome (71% vs. 46% DR4 positive; X21 = 3.97). Toxic complications of RA associated with gold salt treatment are thought to be immunologically mediated [19, 20]. In our study, nine cases of proteinuria, 16 of cutaneous intolerance, three cases of stomatitis, and one case each of leukopenia and hyperthermia were noted. There is a significant excess of DR3- positive patients amongst those with adverse reactions (47% vs. 14% DR3 positive: X21 = 8.96). HLA-DR phenotypes are not significantly correlated with sex, response to corticosteroid therapy, Steinbrocker's radiological stages, gamma globulins, plasma levels of RF, and the presence of antinuclear antibodies. DISCUSSION HLA-DR4 is the only antigen that has been consistently reported to have an association with RA in Caucasians, as first published by Stastny [1]. Negative associations with DR2, DR5, DRw6, and DR7 have been described [4, 7]. This study confirms the high prevalence of the B lymphocyte alloantigen, DR4, and negative associations in RA, of DR5, DRw6, and DR7. In previous studies, risk estimates have been obtained for each antigen relative to all others pooled, and therefore the results are not directly comparable with those presented here. Because of the strong association between DR4 and RA, differences in the risks associated with other antigens may be hidden if DR4 is pooled with the others when obtaining estimates and test statistics. The importance of using a single reference genotype or phenotype for risk calculations has been emphasized by Falk and Rubenstein [21 ] and Svejgaard and Ryder [22]. Although our risk estimates have been obtained under a specific genetic hypothesis, the results include those that may be obtained using contingency table analysis with genotype risks determined by multiplicative allelic risks for d = 0, and additive allelic risks for d = 1. In our data, the estimated risk is highest for the DR414 genotype, but some caution is needed when interpreting this result because of the small number of genotyped DR4 patients. Direct comparisons of the risks without assuming a model of genetic susceptibility, using methods similar to those described in [23], show that the fourfold estimated difference for the DR41 4 and DR41Y genotypes is not significant in this small sample. Differences for the other comparisons of risks from table 6 are significant except for those of the DR41X and DRYIY genotypes. Increased risks associated with particular HLA-DR genotypes have been reported for DR314 in juvenile-type insulin-dependent diabetes mellitus [24], and DR317 in gluten intolerance [25]. Homozygotes carrying the associated antigen appear

HLA-DR GENOTYPE IN RHEUMATOID ARTHRITIS to have a smaller risk of disease than do heterozygotes. In contrast, our results suggest that DR414 homozygotes may have a higher risk of developing RA than DR4 heterozygotes. Further data are required to confirm this hypothesis. The genotype risks in our data are compatible with the inheritance of a single, linked genetic determinant of disease susceptibility, but they do not provide conclusive evidence of the mode of inheritance. We cannot reject the hypothesis of either dominant or recessive inheritance of a possible susceptibility allele. Multiple-case family studies have also given inconclusive results regarding the nature of inheritance of disease susceptibility. Kahn et al. [10] report that of five affected sib-pairs studied, one pair was HLA identical, while the other four shared one parental haplotype. In a second series, Stastny [7] found that affected siblings almost always share one common haplotype. However, in a study of five multicase sibships, Strom and Moller [9] found no increased sharing of haplotypes in affected siblings. Clinical differences correlated with HLA-DR antigens raise the possibility of heterogeneity in disease etiology. Our data confirm the suggestion made at the 7th International Histocompatibility Workshop [6] that the presence of HLA- DR4 is associated with early onset of RA. In some investigations, DR4 has been found to be associated with poor disease prognosis [15, 16], and DR2 with a favorable response to treatment [17]. DR414 homozygotes have been reported to have particularly severe disease [18]. Other studies have found no relationship between disease severity and HLA antigens [7, 26, 27]. In our data, HLA-DR3 is associated with an increased frequency of toxic reactions to sodium aurothiopropanol sulfonate, possibly mediated by immunological factors [19, 20]. The association of high RF titres and DR3 as reported in [19], or DR4 as reported in [7] and [26], was not found in this series of patients. Other clinical variables are not correlated with HLA-DR status in the patients examined here. Alternatively, or in addition to a susceptibility locus linked to the HLA region, HLA-DR4 or other DR antigens might also play a direct role in susceptibility to environmental factors that could initiate the disease process. Epstein-Barr virus (EBV) has been implicated as a possible pathogen in RA [28], and specific T- cell defects restricted to absence of suppression of EBV-induced lymphocyte proliferation have been described in RA patients [29, 30]. Conclusions regarding the genetic nature of susceptibility must be treated with caution while awaiting further evidence regarding the nature of the immunological mechanisms and environmental agents underlying RA. REFERENCES 697 1. STASTNY P: Mixed lymphocyte culture typing cells from patients with rheumatoid arthritis. Tissue Antigens 4:571-591, 1974 2. MCMICHAEL AJ, SASAZUKI T, MCDEVITT HO, PAYNE RO: Increased frequency of HLA-Cw3 and HLA-Dw4 in rheumatoid arthritis. Arthritis Rheum 20:1037-1042, 1977 3. THOMSEN M, MORLING N, SNORRASON E, SVEJGAARD A, S0RENSEN SF: HLA-Dw4 and rheumatoid arthritis. Tissue Antigens 13:56-60, 1979 4. PANAYAI GS, WOOLEY PH: B-lymphocyte allo-antigens in the study of the genetic basis of rheumatoid arthritis. Ann Rheum Dis 36:365-368, 1977

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HLA-DR GENOTYPE IN RHEUMATOID ARTHRITIS 699 28. BRITTON S: EBV and rheumatoid arthritis. Immunol Today 5:127-128, 1982 29. TOSATO G, STEINBERG A, BLAESE M: Defective EBV specific suppressor T-cell function in rheumatoid arthritis. N Engl J Med 305:1238-1243, 1981 30. DEPPER J, BLUESTEIN H, ZVAIFLER N: Impaired regulation of Epstein-Barr virusinduced lymphocyte proliferation in rheumatoid arthritis is due to a T cell defect. J Immunol 127:1899-1902, 1981