A specific HLA-DPB allele is associated with pauciarticular juvenile rheumatoid arthritis but not adult rheumatoid arthritis ( autoimmunity / polymerase chain reaction / sequence-specific oligonucleotide )

Nonradioactive sequence-specific oligonucleotide probes specific for the HLA-DPf3 locus have been used in a simple dot-blot format to type samples amplified by the polymerase chain reaction from 44 patients with pauciarticular juvenile rheumatoid arthritis, 32 patients with adult rheumatoid arthritis, and 50 random controls. The sequences of four new DPfi alleles derived from these patients and controls are reported, bringing the total number of alleles identified thus far to 19. The DPB2.1 allele is significantly increased in juvenile rheumatoid arthritis patients over controls; this allele is not increased in patients with adult rheumatoid arthritis. The association of juvenile rheumatoid arthritis with the DPB2.1 allele is independent of linkage with previously dermed HLA-D region markers of disease. Analysis of the DPB2.1 sequence shows that it differs from the nonsusceptible DPB4.2 allele by only 1 amino acid at position 69 in the I1 domain. A striking association exists between susceptibility to certain human autoimmune disorders and particular alleles of the HLA class II (HLA-D) region (1). This region encodes three related class II antigens, HLA-DR, -DQ, and -DP, which are cell-surface glycoproteins composed ofan a and a P chain (2). With the exception of the DRa and DPa chains, these proteins are highly polymorphic, with the variability localized to the NH2-terminal extracellular domain encoded by the second exon. The role of the class II molecule in the immune response is twofold. (i) Class II molecules present on the stromal cells of the thymus help determine the specificity of the mature T-cell repertoire (3-6). (ii) Class II antigens on the cell surface of antigen-presenting cells bind processed antigen and present it to CD4+CD8T cells, which can become activated and initiate an immune response (7-11). Consequently, an individual's ability to respond to an antigen, whether the antigen be foreign or self, is partly determined by the amino acid sequences of its class II molecules. Indeed, susceptibility of certain autoimmune disorders has been associated with particular class II allelic sequences. For instance, the charge of the amino acid at position 57 in the DQP chain appears to correlate with susceptibility to insulindependent diabetes mellitus (12-14), whereas genetic susceptibility to Pemphigus vulgaris, an autoimmune disease of the skin, appears associated with sequences in both the DRBI locus (DR4-associated) and the DQ/3 locus (DRw6-associated) (15-17). The HLA-DP locus, as defined by the primed lymphocyte typing (PLT) method, a complicated cellular assay, and restriction fragment length polymorphism analyses, has also been implicated in the susceptibility to certain autoimmune diseases (18-22). One such disease is pauciarticular juvenile rheumatoid arthritis (JRA), a type of childhood arthritis of unknown etiology that primarily affects young females, begins with involvement of fewer than five joints, and is associated with antinuclear antibodies in the serum and with inflammatory anterior uveitis. Previous results showed susceptibility to this form of JRA to be associated with an increased frequency of the HLA serotypes DR5, DRw6, and DRw8 (23-28) as well as with the cellularly defined HLA-DP antigen DPw2 (18, 19). Recently, the polymerase chain reaction method was used to amplify the polymorphic second exons of DPa and DP,8 from 34 DPw-typed cell lines (29). Sequence analysis identified 14 DPB alleles and 2 DPa alleles; this is in contrast to the 6 DPw specificities defined by the PLT method. In addition to suggesting that the cellularly defined specificities, DPwl-DPw6, are determined by polymorphic residues within the 8 chain, these data also showed that the DPw2 specificity includes 2DPB3 alleles, the more frequent DPB2.1 allele, and the rarer DPB2.2 allele. These two alleles differ by 3 amino acids in the exon encoding the polymorphic f31 domain (Fig. 1). Based on the 14 DPP sequences, a DPf3 typing system was developed using nonradioactive DP,3 sequence-specific ohgonucleotides as probes in a simple dot-blot format to analyze DNA amplified from the second exon of the DP,8 locus (29, 30). Here, we report the use of this assay to DP,3-type patients with pauciarticular JRA and classical rheumatoid factor-positive adult rheumatoid arthritis. MATERIALS AND METHODS Patients. The 44 pauciarticular JRA patients all meet the standard criteria for this diagnosis, including onset before the age of 16 years of a chronic inflammatory arthritis of at least 6 weeks duration, with fewer than five joints involved in the first 6 months of disease. All 32 adult patients meet the criteria for classic rheumatoid arthritis and, in addition, were positive for serum rheumatoid factor. DP. Typing. DPf3 typing was done by using sequencespecific probes in a dot-blot format to analyze DNA (prepared according to standard procedures; ref. 31) polymerase chain reaction-amplified (32-34) from the second exon of the DPI3 locus. Briefly, the primers UG19 (5'-GCTGCAGGAGAGTGGCGCCTCCGCTCAT-3') and UG21 (5'-CGGATCCGGCCCAAAGCCCTCACTC-3') were used in a 200-1.l reaction to amplify 0.5 tug of DNA for 35 cycles (denaturation: 960C for 30 sec; annealing and extension: 650C for 45 sec). [Note that these primers, in conjunction with this amplification profile, give a much better yield of the polymerase chain reaction product than those described (29). ] Abbreviations: JRA, juvenile rheumatoid arthritis; PLT, primed lymphocyte typing. tTo whom reprint requests should be addressed. 9489 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 9490 Immunology: Begovich et al. DPB4. 1 DPB4. 2 DPB2. 1 DPB2.2 DPO8 oPS DP87 DP83 DPB6 DPB11 DPB1 3 DPOl DPOl. DP89 DPO12 DPB1 4 DPB 15 DOPl 6 DPB1 7 10 20 30 40 50 60 70 Be 90 NYLFQGRQECYAfNGTQRFLERY IYNREEFARFDSOVGEFRAVTELGRPAAEYNSQKDI LEEKRAVPORMCRH1YELGGPMTLOI V DE V DE E LV -E V DE C VADVLV V DEAV-VY-L V DED L DEAD VY-L V DEDL-E DEAVVY-L Q Y L R DEAVVY-L Y C I CAVVY V DEV VA-L V DE E VEAVVl-L V DED E V DEAVVH-L G S V D I-. V-tVH-L V DED L VAV VY QY L R V V DE E DOAVValU -DE EVA B DPw4 DPw4 DPw2 DPw2 DP b I ank DPwS OP blank DPw3 DO6 OP blank OP blank DPwI DP blank OP blank OP blank