HLA-DPAI AND HLA-DPBl IN RHEUMATOID ARTHRITIS AND ITS SUBSETS

The aim of this study was to examine the relationship between HLA-DP and susceptibility to articular and extra-articular features (Felty's syndrome and vasculitis) of rheumatoid arthritis (RA). The possible association of DP types with severity of articular disease was also analysed. No statistically significant associations were observed between HLA-DP alleles and articular or extra-articular features of RA, or to the severity of the arthritis when p was corrected for the number of alleles tested.


INTRODUCTION
Rheumatoid arthritis is a chronic inflammatory disease of unknown aetiology and a genetic predisposition.The strongest genetic association in rheumatoid arthritis has been with HLA-DR4.It has been suggested that the molecular basis for this HLA association is a particular sequence in the third hypervariable region of the DRB I gene (Gregersen et al., 1987).In previous studies we and others have found associations between HLA variants outside the DRBI locus with certain extra-articular features of rheumatoid disease.Thus Felty's syndrome, which is found in less than I per cent of rheumatoid arthritis patients attending rheumatology clinics, has an association at the DQB locus with DQBI*0301 (So et al., 1988;Clarkson et al., 1990), whereas rheumatoid vasculitis shows associations with unusual DQ-DR haplotypic combinations (Hillarby et al., 1993).These associations, which were not accounted for by a primary association at the HLA-DRBllocus with HLA-DR4, suggested that HLA variants outside the DRB 1 locus influence susceptibility to particular rheumatoid disease features.HLA-DQA and HLA-DQB loci map centromeric to HLA-DR loci and it would now be of interest to see whether similar associations may be demonstrated at other HLA loci centromeric to DR.
Few investigations into RA have demonstrated a disease association with HLA-DP (Pawelec et al., 1988;Stephens et al., 1989;Begovich et al., 1989;Perdriger et al., 1992).Until recently this was due to the restrictions of the primed lymphocyte typing (PLT) system.Allelic polymorphism of the DP region has now been studied by RFLP analysis (Stephens et at., 1989) and PCR-SSO DNA dot blot hybridizations (Perdriger et al., 1992) with DPA I and DPB I probes.However, using the method of cellular typing it has been reported that there is a significant decrease in the frequency of DPw3 and a tendential decrease of DPwl in RA (Pawelec et at., 1988), but these decreases do not reach significance when corrected for the number of alleles tested.In Stephens' study DPw4 was increased in RA (87• 5 per cent in RA vs 80•7 per cent in controls), but this difference was not significant when p was corrected.A similar increase was reported in Pawelec's study (79 per cent in RA vs 71 per cent in controls).These methods did not take into account the heterogeneity of the DPw4 specificity.More recently, a DP study using PCR-SSO typing with a panel of 14 probes (Perdriger et at., 1992) revealed an increase of DPB I *040 I in RA (77-46 per cent in RA vs 55•4 per cent in controls) .Gao et at. (1991) have also demonstrated an increase in DPBI *0401, but Begovich et al. (1989) found no association with this DP polymorphism in their adult patient group.However, the relative risk values in both studies (Perdriger et al., 1992;Gao et at., 1991) were relatively low (2•74 and 3•1, respectively).In this report we have examined by PCR-SSO typing, allele frequencies of DPB I and DPA I genes in RA patients with or without extraarticular disease features.In a previous study we demonstrated different HLA associations between rheumatoid subjects with mild and severe articular disease (McMahon et al., 1992).We have therefore examined such groups in this study.

Subjects
All individuals examined were Caucasoid and living in the northwest of England.Seventy unrelated normal subjects were included as controls for DPA I typing and 65 were included as controls for DPB I typing.One hundred and forty-three URrelated patients with classical or definite RA (Ropes, 1959) were DPA I typed and 96 were DPB I typed.None of these patients had Felty's syndrome or major vasculitis.Thirty RA patients with Felty's syndrome were typed for DPA I and 29 for DPB I; Felty's syndrome was defined as RA plus the following; splenomegaly documented by a clinician or ultrasound; white blood cell counts less than 3•5 x 10 9 1-1 and granulocyte counts less than 2•0 x 10 9 I-lover periods of at least 6 months and which were not attributable to drug toxicity.Forty-one subjects with RA and major vasculitis were typed for DPA I and 24 for DPB I. Criteria for major vasculitis were one or more of the following: major cutaneous ulceration, non-arteriosclerotic arterial involvement, scleritis or mononeuritis multiplex.
Articular disease severity in the rheumatoid subjects was assessed by radiological scoring of the hands and feet (Larsen et al., 1915).The relationship between radiographic scores and disease duration has been investigated previously by regression analysis (McMahon et al., 1991), and confidence intervals around a similarly developed regression equation allowed identification of those rheumatoid subjects whose radiographic score and disease duration were in the upper 20 per cent and lower 20 per cent of severity assessments.These patients were classed as 'severe' and 'mild', respectively.Twenty-two severe RA subjects and 29 mild RA subjects were included in the study in order to investigate the relationship of DP with severity of disease.The control group and the RA only group were divided into 'epitope positive' (those who type for the ORB I shared epitope) and 'epitope negative' (those who do not type for this DR sequence).The RA group was divided into seronegative (seronegative was classified as the rheumatoid factor titre being consistently lower than I in 32) and seropositive subsets.
PCR was carried out on I Ilg of genomic DNA by using 30 cycles of amplification.Each cycle comprised three steps: denaturation at 95° C for 1 min, primer annealing at 63° C for 2 min, and finally synthesis of complementary sequences at 72° C for I min.In each reaction mix 2•5 units ofTaq polymerase was used.Amplified DNA was denatured at 95° C for 5 min, cooled on ice, diluted with an equal volume of 20 x SSC and transferred to positively charged nylon membrane (Boehringer Mannheim) using an A TTO dot blotter.Filters were hybridized to one of the 19 DPB specific oligonucleotide probes, or to one of the two DPA specific oligonucleotide probes (Table I).Prehybridization was carried out for 30 min in 5 x SSC, 2 per cent (w/v) blocking reagent (Boehringer Mannheim), 0•1 per cent (w/v) N-Iauroylsarcosine, and 0•02 per cent (w/v) sodium dodecyl sulphate (SDS) at 42° C. Hybridization was in fresh buffer plus probe at 42° C for 18 h in a shaking water bath.DPB I oligonucleotide probes were labelled with digoxigenin and detected with AMPPD according to the manufacturer's instructions (Boehringer Mannheim).
OPAl oligonucleotide probes were radiolabelled with 32p by polynucleotide kinase.Post-hybridization washes were carried out in 6 x SSC for 5 min at room temperature, followed by a further wash in 2 x SSCjO•2 per cent (w/v) SDS at the specified wash temperature (refer to Table I).To validate the specificity of the hybridization and post-hybridization wash conditions reference cell lines were included in each analysis.

Statistics
Differences between DPAI and DPBI frequencies in patients and controls were analysed by (2 x 2) X 2 (with Yates' correction) and Fisher's exact tests.The p values obtained were multiplied by the number of comparisons made to correct for that number of alleles tested (PC).Odds ratios (O.R.) and 95% confidence limits were calculated for each comparison (according to the Mantel-Haenzel method), in order to give a measure of the strength of the association.

RESULTS
The frequencies for DPAI alleles are given in Table 2 and those for DPBl alleles in Tables 3 and 4. DPAI *01 is the most frequent DPAI allele in all the subject groups studied (90 per cent in the control group and 99 per cent in the RA group), whereas the frequency of DPAI *0201 is lower (40 per cent in controls and 34 per cent in RA; Table 2).Among the different DPBl alleles, DPBl *0401 has the highest frequency (54 per cent in controls, 58 per cent in RA, 38 per cent in RA-vasculitis, and 69 per cent in RA-Felty's), followed by DPBl *0402 (23 per cent in controls, 27 per cent in RA, 25 per cent in RA-vasculitis, and 35 per cent in RA-Felty's; Table 3).DPBI*0101 and 0201 were moderately frequent (25 per cent and 20 per cent in controls and 15 per cent and 13 per cent in RA, respectively).Particular alleles were infrequently present; these being DPBI *0202,0601,0901, and 1101.
No association between DPAI or DPBI alleles and severity of disease was detected (Table 3) or with the epitope negative or epitope positive patients (Table 4).

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Results for DPBl typing in seronegative subjects are presented in Table 5; as only seven seronegatives had been typed no comparisons were made with seropositive subjects.

DISCUSSION
In this study we found DPAI *01 and DPAI *0201 control allele frequencies are in agreement with previous observations (AI-Daccak et al., 1991) and there are no significant associations between DPAI and any of the disease groups studied.
Cellular typing ofDP alleles has previously demonstrated a decrease in the DPw3 allele in RA subjects versus controls (Pawelec et al., 1988), however our study shows that the DPBI *0301 gene frequency is practically the same in both groups (5 per cent in controls and 4 per cent in RA).It has previously been reported that DPB I *0201 is significantly increased in juvenile RA patients over controls, but not increased in patients with adult RA (Begovich et al., 1989) and our results are concordant with this adult RA study.This study did not detect the reported increase ofDPB I *0301 in seronegative patients (Gao et al. , 1991), however only seven seronegative subjects were included in our RA population.No significant associations were detected with the severity of erosive disease in RA.
Cellular typing ofDP alleles has previously shown a decrease of the DPw I allele in Felty's syndrome (Pawelec et al. , 1988).Our study also demonstrated a decrease in DPB I *0 I 0 I in Felty's syndrome.This was not statistically significant when p was corrected for the number of alleles tested and it should be taken into consideration that only 29 cases of Felty's syndrome were DPB typed in our study.This study has demonstrated that there is a lack of association between OPAl or DPBI alleles and disease.Hence, there is no evidence to suggest that HLA-DP alleles have a primary effect on susceptibility to RA.This includes subjects that do not type for the known DR susceptibility gene.These results also suggest that DP alleles do not increase the risk of extra-articular disease features (except for RA-vasculitis, where statistically non-significant increases for alleles OPBI *0801, 1001, and 1801 and decrease for allele OPBI *0401 were observed).Additionally, there is no indication that either OPBI or OPAl alleles are associated with severity of arthritis in rheumatoid disease.However, larger rheumatoid disease subset groups will be necessary (to overcome the difficulties and limitations encountered when small numbers of patients are involved) in order to provide 'significant' evidence.

Table 1 .
DPA and DPB SSO probe sequences and wash temperatures