European Journal of Immunogenetics (1991), 18,259-263
SHORT COMMUNICATION
H L A D P B l ALLELES A N D SUSCEPTIBILITY T O RHEUMATOID ARTHRITIS D . A . S A G E ,P . R . E V A N S ,M . I . D . C A W L E Y ,* J . L . S M I T H& W . M . H O W E L L Wessex Histocompatibility Group and * Rheumatology Unit, Southampton General Hospital, Southampton, U. K . (Received 11 March 1991; accepted 26 March 1991)
SUMMARY
HLA-DPB1 genotypic and phenotypic frequencies were investigated in a series of 35 adult rheumatoid arthritis (RA) patients and 42 controls. No significant associations between DPBl alleles and susceptibility to R A were demonstrated, although some non-significant differences in DPBl*0301 and 0401 allele frequencies between patients and controls were observed.
There is a well-documented association between HLA-DR4 and susceptibility to rheumatoid arthritis (RA) in most ethnic groups (Tiwari & Terasaki, 1985), with increases in DR4 Dw4 and Dw14 ceilularly defined subtypes reported in some studies (Nepom et al., 1987; Wordsworth et a f . ,1989). A secondary association between DR1 and RA susceptibility has also been observed in some populations (Winchester, 1986; Howell et af., 1989). Sequencing studies have identified common epitopes on the DRPl chains of DR4 and DR1 haplotypes (Wordsworth et al., 1989), suggesting a shared epitope hypothesis for susceptibility to this disease. Evidence for D Q involvement has also been reported in certain restriction fragment length polymorphism (RFLP)-based studies (Sansom et al., 1987; Howell et al., 1989). However, there have been few studies on the possible contribution of DP alleles to R A susceptibility due to limitations in D P typing by the primed lymphocyte test (PLT) (Wank & Schendel, 1984) and by DNA-RFLP analysis (Bodmer et al., 1987), since both techniques are cumbersome and as yet are unable to identify all known DPBl alleles (Bodmer et al., 1990). Nevertheless it is important to investigate the role of DP in susceptibility to R A since the DP gene product is known to be expressed at high levels in the rheumatoid synovium (Teyton et al., 1987; Walters et al. , 1987) and this expression is significantly reduced following treatment with gold or penicillamine (Walters et a f . , 1987). The limited studies relating to HLA-DP in RA report conflicting data: PLT-based studies have reported a possible ‘protective’ role for DPB1*0301 in RA susceptibility (Pawelec et al., 1988). Correspondence:D.A. Sage, Wessex HistocompatibilityGroup, Regional Immunology Service, Tenovus Laboratory, Southampton General Hospital, Tremona Road, Southampton SO9 4XY, U.K.
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Conversely, RFLP studies have demonstrated an increase in DPB1*04 in RA patients (Stephens et al., 1989). A recent report using polymerase chain reaction and sequence-specific oligonucleotide probing (PCR-SSOP) has identified no association between DPBl alleles and susceptibility to adult onset R A , although an increase in DPB1*0201 was observed amongst pauciarticularjuvenile RA patients (Begovich et al., 1989). It is yet not clear whether these associations are independent of DWDQ-DP genetic linkage, although another study reporting an increase demonstrated that this is dependent upon linkage to DR5 (Van Kerckhove et al., 1990). In this preliminary study we have used PCR-SSOP typing to investigate possible associations between HLA-DPB1 alleles and susceptibility or resistance to adult onset RA in the British population. PCR-SSOP typing was used due to its superior sensitivity. In addition, in other autoimmune diseases such as multiple sclerosis, possible DP associations detected by PLT (Odum et a l . , 1988) have not been supported by subsequent PCR-SSOP baseed studies, even in the same ethnic group (Begovich et al., 1990). Thirty-five patients attending the Southampton Rheumatology Unit (17 male, 18 female) with established classical or definite and seropositive RA without Feltys syndrome and 42 controls randomly chosen from the Wessex Bone Marrow Donor panel and from among cadaveric organ donors were included in the study. All patients and controls were of Caucasian ethnic origin. The phenotypic frequencies of DR4 and DR1 in the panel of RA patients from which this series was drawn were 70% and 26%, respectively (Howell et al., 1989), compared with 36% and 16% for the U.K. population as a whole (Klouda et at., 1985). PCR gene amplification of the second exon of the DPBl gene was performed as described previously (Howell et al., 1991a), using 1-pg aliquots of DNA, 2.5 units DNA Taq Polymerase (Perkin Elmer, Beaconsfield, Bucks, U.K. and Promega, Southampton, Hants, U.K.) and 1p~ of each primer, for 35 cycles. After the initial denaturing step, all cycles consisted of 2 min at 94"C, 2 min at 50°C and 2 min at 72°C. Two-microlitre aliquots of amplified product were dot-blotted onto Hybond N nylon filters (Amersham International, Amersham, Bucks, U.K.), UV crosslinked, prehybridized and hybridized using 5 pmoles of each a3'P-3' end-labelled oligonucleotide probe. Positive hybridizations were visualized with Fuji-RX film after 1-24 h exposure at -70°C. Filters were dehybridized and sequentially hybridized with a panel of 15 oligonucleotide probes. This method detects 17 out of 19 WHO-defined DPBl alleles (Bodmer et al., 1990). Alleles DPB1*080! and 0001 show identical hybridization patterns to DPBl*1601 and 1701, respectively. These alleles can be distinguished using additional probes. This typing system has been described in detail elsewhere (Howell et al., 1991a). Differences between DPBl phenotypic and genotypic frequencies in patients and controls were analysed by the xzand Fisher's exact tests. The Fisher's exact test provided a more accurate estimate of P value when allele numbers for either patients or controls were less than 5 . The P values obtaincd were multiplied by the number of comparisons made (i.e. 17) to correct for the number of alleles tested (Batchelor & McMichael, 1987). Analyses were carried out by 2 x 2 contingency tables using the University of Southampton Faculty of Medicine MEDSTAT package. Phenotypic and genotypic frequencies for RA patients and controls are shown in Tzble 1. No statistically significant differences were seen in either phenotypic or genotypic frequencies for any allele, using either x2 or Fisher's exact tests at P = 0.05, correcting for the number of alleles tested for. DPB1*0401 was the most frequent allele in both RA patients and controls. Control phenotypic frequencies were in agreement with those seen in U.S. Caucasian (Fernandez-Vina et al., 1991) and French Canadian Caucasian populations (Howell et al., 1991b). Genotypic frequencies were also consistent with those reported in U.S. and British Caucasian populations (Begovich et al.,
HLA DPBl alleles and susceptibility to RA
261
TABLE 1. DPBl phenotypic and genotypic frequencies in adult RA patients and controls Genotypic frequency (YO) DPBl allele 0101 0201 0202 0301 * 0401t 0402 0501 0601 080111601 0901/1701 1001 1101 1301 1401 1501 1801 1901
Phenotypic frequency (%)
RA patients (nl = 70)
Controls (nl = 84)
RA patients (n2= 35)
(nz= 42)
4(3) 16(11) W) 6(4) 54(38) 7(5) O.O(O) O.O(O) O.O(O) 1(1) O.O(O) W) 4(3) 3(2) 1(1) O.O(O) O.O(O)
5(4) 12( 10) O.O(O) 15(13) 39(33) 12(10) 5(4) 2(2) W) O.O(O) 1(1) 4(3) 4(3) O.O(O) O.O(O) O.O(O) O.O(O)
9(3) 26(9) 3(11 11(4) 74(26) 14(5) O.O(O) O.O(O) O.O(O) 3(1) O.O(O) 3(1) 9(3) 6(2) 3(1) O.O(O) O.O(O)
N4) 19(8) 0.0(0) 26( 11) 57(24) 24(10) 10(4) 5(2) 20) O.O(O) 20) 7(3) 7(3) O.O(O) O.O(O) O.O(O) O.O(O)
Controls
All differences were non-significant at the P = 0.05 level, correcting for the number of alleles tested. n, = number of RA or control chromosomes. n2 = number of RA patients or controls. *Uncorrected P = 0.09; relative risk (RR) = 0.3 (based on genotypic frequencies). tuncorrected P = 0.09; relative risk (RR) = 1.8 (based on genotypic frequencies).
1990; Howell ef al., 1991a). These data are valuable in further establishing the frequencies of DPBl phenotypes and genoiypes in Caucasian populations. Although no significant differences in DPBl allele frequencies were observed between RA patients and controls, some non-significant differences -:'ere detected, the most pronounced being for DPB1*0301 and 0401. DPB1*0301 was decreased in RA patients compared with controls. This difference was more pronounced at the phenotypic level (11% versus 29%). Although this difference was not significant in this preliminary study, it is of note that Pawelec et al. (1988) demonstrated a significant negative association between DPB1*0301 and RA in a series of 111 patients and 272 controls, based on PLT typing. Stephens et al. (1989), using RFLP analysis, also reported a significantly lower frequency of the DPBl*0301/0601 genotype in DR4-positive RA patients, indicating that the DPB1*0301/0601 genotypes may confer some degree of protection when combined with the major HLA class I1 susceptibility allele for RA. In the latter study DPB1'0301 and DPBl*O601 frequencies were combined since it was not possible to distinguish them using the typing system in question. In contrast, a recent report by Gao et al. (1990) demonstrated that DPB1*0301 is a major risk factor in rheumatoid factor negative adult RA. In our study all patients were rheumatoid factor positive. These findings may indicate a role for the DPB1*0301 allele either in increasing susceptibiiity to or having a 'protective' effect in different subsets of adult onset RA, as manifested by the presence or absence of rheumatoid factor.
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A slight increase in the frequency of DPBl*O401 in RA patients compared with controls was observed (74% versus 63% at the phenotypic level). Stephens et al. (1989) also reported a non-significant increase in DPBl*0401 amongst RA patients and concluded that this was independent of linkage to DR4 or DR1. This is in conflict with the findings of Gao et al. (1990) who reported linkage between DR4 and DPBl *O401. However, the only well-documented DR-DP linkage is between DR3 and DPB1*0101 (Sanchez-Perez & Shaw, 1986). These preliminary data suggest that it is unlikely that DPBl alleles play a highly significant independent role in determining susceptibility or resistance to RA. However, they d o confirm a possible minor role for DPB1*0301. in agreement with earlier studies (Pawelec et al., 1988; Stephens et al., 1989; Gao ef al., 1990). An independent role for DPB1*0401 remains equivocal. We conclude that further studies are required to determine the precise role of DPBl alleles in susceptibility to R A and whether such associations are dependent upon, or independent of, DWDQ and D P linkages in the ethnic groups in question.
REFERENCES BATECHLOR, J . R . & MCMICHAEL. A.J. ( 1987) Progress in understanding HLA and disease associations. British Medical Bulletin. 43, 156. BEGOVICH.A.B., BUGAWAN, T.L.. NEPOM, B.S., KLITZ,W., NEPOM. G.T. & ERLICH,H.A. (1989) A specific HLA-DPP allele is associated with pauciarticular juvenile rheumatoid arthritis but not adult rheumatoid arthritis. Proceedings of the National Academy of Sciences, U . S . A . ,86, Y489. BEGOVICH, A.B., HELMUTH. R.C., OKSENBUKG. J .R.. SAKAI. K . , TABIRA. T . , SASAZUKI. T . , STEINMANN. L. & ERLICH. H.A. (1990) HLA DPB and susceptibility to multiple sclerosis: an analysis of Caucasoid and Japanese patient populations. Human Immunology:v.28, 365. BODMER, J., BODMER, W., HEYES,J . So, A , , TCINKS. S . , TROWSI)AI.F., J . & YOUNG, J. (1987) Identification of HLA-DP polymorphism with D P a and DPP probes and monoclonal antibodies: correlation with primed lymphocyte typing. Proceedings of the National Academy of Sciences, U . S . A . , 84, 4596. BODMER,J . , MARSH,S.G.E. & ALBERT,E . (1990) Nomenclature for factors of the HLA system, 1989. Immunology Today, 11,3. FERNANDEZ-VINA, M., MORAES.M.E. & STASTNY, P. (1991) DNA typing for class I1 HLA antigens with allele specific or group specific amplification. 111. Typing for 24 alleles of HLA-DP. Human Immunology, 30,60. GAO,X . , FERNANDEZ-VINA, M., OLSEN,N., PINTUS, T. & STASTNY. P. (1990) DPB1*0301 is a major risk factor for rheumatoid factor negative adult rheumatoid arthritis. Human Immunology. Sixteenth Annual ASHI meeting supplement. HOWELL,W.M., EVANS, P.R., WILSON,P.J., CAWLEY, M.I.D. & SMITH, J.L. (1989) HLA class I1 DR. DQ and D P restriction fragment length polymorphisms in rheumatoid arthritis. Annals of the Rheumatic Diseases, 48,295. HOWELL,W.M., SAGE,D.A., HAEGERT.D.G., EVANS,P.R. & SMITH.J.L. (1991a) PCR-SSO typing for HLA-DPB alleles. European Journal oflrnmunogenetics, 18, 81. HOWELL,W.M., SAGE,D.A., EVANS,P . R . , SMITH,J.L., FRANCIS, G.S. & HAEGERT, D.G. (1991b) No association between susceptibility to multiple sclerosis and HLA-DPBl alleles in the French Canadian population. Tissue Antigens, 37, 156. KLOUDA,P.T., MOLNAR, J., WASIK,A. & BODMER, J. (1985) HLA-A, B and D R antigens in the British Isles. U.K . Transplant Service Report. NEPOM,G.T., HANSEN,J.A. & NEPOM,B.S. (1987) The molecular basis for HLA class I1 associations with rheumatoid arthritis. Journal of Clinical Immunology, 7 , 1. ODUM,N., HYLDIG-NIELSEN, J.J., MORLING, N., SANDBERG-WOLLHEIM, M., PLATZ,P. & SVEJGAARD, A. (1988) HLA-DP antigens are involved in the susceptibility to multiple sclerosis. Tissue Antigens, 31,235. PAWELEC,G., REEKERS, P., BRACKERTZ, D . , SANSOM. D . , SCHNEIDER, E.M., BLAUROCK, M., MULLER,C., REHBEIN, A., BALKO,1. & WERNET,P. (1988) HLA-DP in rheumatoid arthritis. Tissue Antigens, 31,83.
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SANCHEZ-PEREZ, M. & SHAW,S. (1986) HLA-DP: current status. In: HLA Class II Antigens: A Comprehensive Review of Structure and Function (eds B.G. Solheim, E . Moller and S. Ferrone), pp. 8S108. Springer-Verlag. Berlin. SANSOM, D.M., BIDWELL, J.L., MADDISON, P.J., CAMPION, G., KLOUDA,P.T. & BRADLEY, B.A. (1987) HLA DQa and DQp RFLPs associated with Feltys Syndrome and DR4 positive rheumatoid arthritis. Human Immunology, 19, 269. STEPHENS, H.A.F., VAUGHAN, R.W., SAKKAS, L.I., WELSH,K.1. & PANAYI, G.S. (1989) Southern blot analysis of HLA-DP gene polymorphisms in Caucasoid rheumatoid arthritis patients and controls. Immunogenetics, 30, 149. TEYTON,L., LOTEAU, V., TURMEL, P., ARENZANA-SEISDEDOS, F., VIRELIZIER, J., PUJOL,J., LOYAU, G., PIATIER-TONNEAU, D., AUFFRAY,C. & CHARRON, D.J. (1987) HLA DR, DQ and DP antigen expression in rheumatoid synovial cells: A biochemical and quantitative study. Journal of Immunology, 138, 1730. J. & TERASAKI, P. (1985) HLA and Disease Associations. Springer Verlag, New York. TIWARI, VANKERCKHOVE, C., LUYRINK, L., ELMA,M.S., MAKSYMOWYCH, W.P., LEVINSON, J.E., LARSON, M.G., CHOI,E. & GLASS,D.N. (1990) HLA-DP/DR interaction in children with juvenile rheumatoid arthritis. Immunogenetics, 32, 364. M.I.D. (1987) An investigation of the WALTERS, M.T., SMITH, J.L., MOORE,K., EVANS, P.R. & CAWLEY, action of disease modifying antirheumatic drugs on the rheumatoid synovial membrane: reduction in T lymphocyte subpopulations and HLA-DP and DQ antigen expression after gold or penidlamine therapy. Annals of the Rheumatic Diseases, 46, I . WANK,R. & SCHENDEL, D.J. (1084) Genetic analysis of HLA-D region products defined by PL,T. In: Histocompatibility Testing (eds E.D. Albert, M.P. Baur and W.R. Mayr), pp. 289-299. Springer-Verlag, Berlin. WINCHESTER, R.J. (1986) The HLA system and susceptibility to diseases: an interpretation. In: Clinical Aspecrs of Autoimmunity (ed. E. Tan), pp. 9-16. New York Transmedica. WORDSWORTH, B.P., LANCHBURY, J.S.S., SAKKAS, L.I., WELSH,K.I., PANAYI, G.S. & BELL,J.I. (1989) HLA DR4 subtype frequencies in rheumatoid arthritis indicate that DRBl is the major susceptibility locus within the HLA class I1 region. Proceedings ofthe National Academy of Sciences, U.S.A.,86, 10049.
SHORT COMMUNICATION
H L A D P B l ALLELES A N D SUSCEPTIBILITY T O RHEUMATOID ARTHRITIS D . A . S A G E ,P . R . E V A N S ,M . I . D . C A W L E Y ,* J . L . S M I T H& W . M . H O W E L L Wessex Histocompatibility Group and * Rheumatology Unit, Southampton General Hospital, Southampton, U. K . (Received 11 March 1991; accepted 26 March 1991)
SUMMARY
HLA-DPB1 genotypic and phenotypic frequencies were investigated in a series of 35 adult rheumatoid arthritis (RA) patients and 42 controls. No significant associations between DPBl alleles and susceptibility to R A were demonstrated, although some non-significant differences in DPBl*0301 and 0401 allele frequencies between patients and controls were observed.
There is a well-documented association between HLA-DR4 and susceptibility to rheumatoid arthritis (RA) in most ethnic groups (Tiwari & Terasaki, 1985), with increases in DR4 Dw4 and Dw14 ceilularly defined subtypes reported in some studies (Nepom et al., 1987; Wordsworth et a f . ,1989). A secondary association between DR1 and RA susceptibility has also been observed in some populations (Winchester, 1986; Howell et af., 1989). Sequencing studies have identified common epitopes on the DRPl chains of DR4 and DR1 haplotypes (Wordsworth et al., 1989), suggesting a shared epitope hypothesis for susceptibility to this disease. Evidence for D Q involvement has also been reported in certain restriction fragment length polymorphism (RFLP)-based studies (Sansom et al., 1987; Howell et al., 1989). However, there have been few studies on the possible contribution of DP alleles to R A susceptibility due to limitations in D P typing by the primed lymphocyte test (PLT) (Wank & Schendel, 1984) and by DNA-RFLP analysis (Bodmer et al., 1987), since both techniques are cumbersome and as yet are unable to identify all known DPBl alleles (Bodmer et al., 1990). Nevertheless it is important to investigate the role of DP in susceptibility to R A since the DP gene product is known to be expressed at high levels in the rheumatoid synovium (Teyton et al., 1987; Walters et al. , 1987) and this expression is significantly reduced following treatment with gold or penicillamine (Walters et a f . , 1987). The limited studies relating to HLA-DP in RA report conflicting data: PLT-based studies have reported a possible ‘protective’ role for DPB1*0301 in RA susceptibility (Pawelec et al., 1988). Correspondence:D.A. Sage, Wessex HistocompatibilityGroup, Regional Immunology Service, Tenovus Laboratory, Southampton General Hospital, Tremona Road, Southampton SO9 4XY, U.K.
259
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Conversely, RFLP studies have demonstrated an increase in DPB1*04 in RA patients (Stephens et al., 1989). A recent report using polymerase chain reaction and sequence-specific oligonucleotide probing (PCR-SSOP) has identified no association between DPBl alleles and susceptibility to adult onset R A , although an increase in DPB1*0201 was observed amongst pauciarticularjuvenile RA patients (Begovich et al., 1989). It is yet not clear whether these associations are independent of DWDQ-DP genetic linkage, although another study reporting an increase demonstrated that this is dependent upon linkage to DR5 (Van Kerckhove et al., 1990). In this preliminary study we have used PCR-SSOP typing to investigate possible associations between HLA-DPB1 alleles and susceptibility or resistance to adult onset RA in the British population. PCR-SSOP typing was used due to its superior sensitivity. In addition, in other autoimmune diseases such as multiple sclerosis, possible DP associations detected by PLT (Odum et a l . , 1988) have not been supported by subsequent PCR-SSOP baseed studies, even in the same ethnic group (Begovich et al., 1990). Thirty-five patients attending the Southampton Rheumatology Unit (17 male, 18 female) with established classical or definite and seropositive RA without Feltys syndrome and 42 controls randomly chosen from the Wessex Bone Marrow Donor panel and from among cadaveric organ donors were included in the study. All patients and controls were of Caucasian ethnic origin. The phenotypic frequencies of DR4 and DR1 in the panel of RA patients from which this series was drawn were 70% and 26%, respectively (Howell et al., 1989), compared with 36% and 16% for the U.K. population as a whole (Klouda et at., 1985). PCR gene amplification of the second exon of the DPBl gene was performed as described previously (Howell et al., 1991a), using 1-pg aliquots of DNA, 2.5 units DNA Taq Polymerase (Perkin Elmer, Beaconsfield, Bucks, U.K. and Promega, Southampton, Hants, U.K.) and 1p~ of each primer, for 35 cycles. After the initial denaturing step, all cycles consisted of 2 min at 94"C, 2 min at 50°C and 2 min at 72°C. Two-microlitre aliquots of amplified product were dot-blotted onto Hybond N nylon filters (Amersham International, Amersham, Bucks, U.K.), UV crosslinked, prehybridized and hybridized using 5 pmoles of each a3'P-3' end-labelled oligonucleotide probe. Positive hybridizations were visualized with Fuji-RX film after 1-24 h exposure at -70°C. Filters were dehybridized and sequentially hybridized with a panel of 15 oligonucleotide probes. This method detects 17 out of 19 WHO-defined DPBl alleles (Bodmer et al., 1990). Alleles DPB1*080! and 0001 show identical hybridization patterns to DPBl*1601 and 1701, respectively. These alleles can be distinguished using additional probes. This typing system has been described in detail elsewhere (Howell et al., 1991a). Differences between DPBl phenotypic and genotypic frequencies in patients and controls were analysed by the xzand Fisher's exact tests. The Fisher's exact test provided a more accurate estimate of P value when allele numbers for either patients or controls were less than 5 . The P values obtaincd were multiplied by the number of comparisons made (i.e. 17) to correct for the number of alleles tested (Batchelor & McMichael, 1987). Analyses were carried out by 2 x 2 contingency tables using the University of Southampton Faculty of Medicine MEDSTAT package. Phenotypic and genotypic frequencies for RA patients and controls are shown in Tzble 1. No statistically significant differences were seen in either phenotypic or genotypic frequencies for any allele, using either x2 or Fisher's exact tests at P = 0.05, correcting for the number of alleles tested for. DPB1*0401 was the most frequent allele in both RA patients and controls. Control phenotypic frequencies were in agreement with those seen in U.S. Caucasian (Fernandez-Vina et al., 1991) and French Canadian Caucasian populations (Howell et al., 1991b). Genotypic frequencies were also consistent with those reported in U.S. and British Caucasian populations (Begovich et al.,
HLA DPBl alleles and susceptibility to RA
261
TABLE 1. DPBl phenotypic and genotypic frequencies in adult RA patients and controls Genotypic frequency (YO) DPBl allele 0101 0201 0202 0301 * 0401t 0402 0501 0601 080111601 0901/1701 1001 1101 1301 1401 1501 1801 1901
Phenotypic frequency (%)
RA patients (nl = 70)
Controls (nl = 84)
RA patients (n2= 35)
(nz= 42)
4(3) 16(11) W) 6(4) 54(38) 7(5) O.O(O) O.O(O) O.O(O) 1(1) O.O(O) W) 4(3) 3(2) 1(1) O.O(O) O.O(O)
5(4) 12( 10) O.O(O) 15(13) 39(33) 12(10) 5(4) 2(2) W) O.O(O) 1(1) 4(3) 4(3) O.O(O) O.O(O) O.O(O) O.O(O)
9(3) 26(9) 3(11 11(4) 74(26) 14(5) O.O(O) O.O(O) O.O(O) 3(1) O.O(O) 3(1) 9(3) 6(2) 3(1) O.O(O) O.O(O)
N4) 19(8) 0.0(0) 26( 11) 57(24) 24(10) 10(4) 5(2) 20) O.O(O) 20) 7(3) 7(3) O.O(O) O.O(O) O.O(O) O.O(O)
Controls
All differences were non-significant at the P = 0.05 level, correcting for the number of alleles tested. n, = number of RA or control chromosomes. n2 = number of RA patients or controls. *Uncorrected P = 0.09; relative risk (RR) = 0.3 (based on genotypic frequencies). tuncorrected P = 0.09; relative risk (RR) = 1.8 (based on genotypic frequencies).
1990; Howell ef al., 1991a). These data are valuable in further establishing the frequencies of DPBl phenotypes and genoiypes in Caucasian populations. Although no significant differences in DPBl allele frequencies were observed between RA patients and controls, some non-significant differences -:'ere detected, the most pronounced being for DPB1*0301 and 0401. DPB1*0301 was decreased in RA patients compared with controls. This difference was more pronounced at the phenotypic level (11% versus 29%). Although this difference was not significant in this preliminary study, it is of note that Pawelec et al. (1988) demonstrated a significant negative association between DPB1*0301 and RA in a series of 111 patients and 272 controls, based on PLT typing. Stephens et al. (1989), using RFLP analysis, also reported a significantly lower frequency of the DPBl*0301/0601 genotype in DR4-positive RA patients, indicating that the DPB1*0301/0601 genotypes may confer some degree of protection when combined with the major HLA class I1 susceptibility allele for RA. In the latter study DPB1'0301 and DPBl*O601 frequencies were combined since it was not possible to distinguish them using the typing system in question. In contrast, a recent report by Gao et al. (1990) demonstrated that DPB1*0301 is a major risk factor in rheumatoid factor negative adult RA. In our study all patients were rheumatoid factor positive. These findings may indicate a role for the DPB1*0301 allele either in increasing susceptibiiity to or having a 'protective' effect in different subsets of adult onset RA, as manifested by the presence or absence of rheumatoid factor.
262
D. A . Sage et al.
A slight increase in the frequency of DPBl*O401 in RA patients compared with controls was observed (74% versus 63% at the phenotypic level). Stephens et al. (1989) also reported a non-significant increase in DPBl*0401 amongst RA patients and concluded that this was independent of linkage to DR4 or DR1. This is in conflict with the findings of Gao et al. (1990) who reported linkage between DR4 and DPBl *O401. However, the only well-documented DR-DP linkage is between DR3 and DPB1*0101 (Sanchez-Perez & Shaw, 1986). These preliminary data suggest that it is unlikely that DPBl alleles play a highly significant independent role in determining susceptibility or resistance to RA. However, they d o confirm a possible minor role for DPB1*0301. in agreement with earlier studies (Pawelec et al., 1988; Stephens et al., 1989; Gao ef al., 1990). An independent role for DPB1*0401 remains equivocal. We conclude that further studies are required to determine the precise role of DPBl alleles in susceptibility to R A and whether such associations are dependent upon, or independent of, DWDQ and D P linkages in the ethnic groups in question.
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