HLA-DRB1 ALLELES ASSOCIATED WITH RHEUMATOID ARTHRITIS IN NORTHERN ITALY: CORRELATION WITH DISEASE SEVERITY

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1 British Journal of Rheumatology 1998;37: HLA-DRB1 ALLELES ASSOCIATED WITH RHEUMATOID ARTHRITIS IN NORTHERN ITALY: CORRELATION WITH DISEASE SEVERITY C. SALVARANI, P. L. MACCHIONI, W. MANTOVANI, M. BRAGLIANI, E. COLLINA, T. CREMONESI,* B. BATTISTEL* and L. BOIARDI Unità Reumatologica and *2a Divisione di Radiologia, Azienda Ospedaliera Arcispedale S. Maria Nuova, Reggio Emilia and Laboratorio Analisi Malpighi, Settore Tipizzazione Tissutale, Azienda Policlinico S. Orsola-Malpighi, Bologna, Italy SUMMARY The aim of the study was to evaluate the relationship between the presence of the rheumatoid epitope, defined by a sequence motif in the HLA-DRB1 alleles, rheumatoid factor and disease severity in Northern Italian patients with rheumatoid arthritis ( RA). Twenty-nine DR4-positive and 57 DR4-negative RA patients were studied. Each DR4-positive patient was matched with two DR4-negative controls of similar disease duration and sex. HLA-DRB1 alleles were determined in the 86 patients and 351 controls from the same geographical area. The patients were retrospectively evaluated for extra-articular features ( EAF ) and radiographic damage. The rheumatoid epitope was expressed in 45% of patients. No significant differences in the presence of rheumatoid factor, EAF and articular damage were observed between patients with no, one or two doses of epitope. However, the patients encoding the epitope by an HLA-DR4 allele had a higher number of eroded joints and a higher Larsen score compared to those without the epitope. No differences were present between patients expressing HLA-DRB1*01 alleles and those lacking the rheumatoid epitope. Even in the absence of expression of the rheumatoid epitope, seropositive patients had more EAF and more erosive disease compared to those who were seronegative. Even if most Northern Italian RA patients do not express the rheumatoid epitope, the radiological severity of disease is associated with HLA-DRB1*04 alleles. KEY WORDS: Rheumatoid arthritis, Severity, HLA-DRB1 alleles, Rheumatoid factor. THE association between HLA class II genes and a the milder disease expression observed in these predisposition to rheumatoid arthritis ( RA) is well populations. established. Many White population studies have In a previous study [4], we observed a weak association shown that RA is associated with the DRB1-encoded with DR4 (relative risk 2.4) in a series of variants HLA-DR4 and DR1. Northern Italian patients with RA. A low frequency DNA sequencing studies have identified a similar of EAF (32%) was present in our patients. A parallel amino acid sequence (QK/RRAA) at residues weak association with DR4 and a low frequency of of the third hypervariable region of the DRB1 EAF were also observed in Greek patients with RA gene, shared by the RA-associated DR4 subtypes [5, 7]. (DRB1*0401, *0404/0408, *0405) as well as by DR1 In the present study, we examined whether the (DRB1*0101, *0102), but not shared by non-ra- rheumatoid epitope is a marker of disease severity in associated DR alleles [1]. The involvement of a group Northern Italian patients with RA. We compared the of related rheumatoid epitopes has been postulated presence of EAF and the severity of radiological as the basis for the HLA association in RA [2]. damage among patients having none, one or two doses Weyand et al. [3] showed that the severity of RA, of the rheumatoid epitope encoded by their respective as defined by extra-articular manifestations ( EAF ) and HLA-DRB1 alleles. We also evaluated disease severity destructive arthritis, is correlated with the dose of the in DR1-positive and DR4-negative patients, and compared rheumatoid epitope. Seropositive patients who inherit EAF frequency and erosive disease between this a disease-linked HLA-DRB1 on both haplotypes group, patients DR4 positive with the rheumatoid develop a more aggressive disease with EAF and epitope and patients negative for DR4 and DR1. The increased frequency of joint surgery. relationship between rheumatoid factor, HLA-DRB1 RA seems to be milder in Mediterranean populations alleles and disease severity was also evaluated. than in Caucasian RA patients originating from Northern Europe [4 6 ]. The frequency of the rheumatoid PATIENTS AND METHODS epitope is higher in Northern European than in Patients and controls Mediterranean RA patients; a low frequency of this We identified 29 HLA-DR4-positive RA patients epitope is also observed in Mediterranean general (disease duration 8.2 ± 7.2 yr) and 57 HLA-DR4- populations [7 9]. In particular, homozygosity for negative RA patients matched for disease duration the DR4 specificity is uncommon in Mediterranean (8.9 ± 6.9 yr) and sex. Each DR4-positive patient was RA patients [6, 8] and such rarity could influence matched with two DR4-negative controls of similar disease duration and sex; for one DR4-positive patient, Submitted 3 February 1997; revised version accepted 19 June only one adequately matched DR4-negative patient Correspondence to: C. Salvarani, Unità Reumatologica, Azienda was available. Ospedaliera Arcispedale S. Maria Nuova, V. le Umberto 1 N 50, These patients represented a series of RA patients Reggio Emilia, Italy. seen as out-patients at the Rheumatological Unit of British Society for Rheumatology

2 166 BRITISH JOURNAL OF RHEUMATOLOGY VOL. 37 NO. 2 Reggio Emilia Hospital (a secondary referral centre) HLA-DNA typing by two of the authors (CS and PM) during a 1 yr Genomic DNA was extracted from whole blood of period (1992). Clinical and demographic data are patients and controls by using a rapid salting-out summarized in Table I. All the patients originating method [13]. from Emilia Romagna (Northern Italy) fulfilled the A low-resolution HLA-DRB1 molecular typing was ACR 1987 criteria for RA [10] and were followed performed by polymerase chain reaction amplification up for at least 1 yr. Medical records of the patients with sequence-specific primers ( PCR-SSP), as previously were reviewed for the presence of rheumatoid described [14]. This method allowed us to type factor (patients were considered seropositive if the 18 different DRB1 molecular alleles, including Waaler Rose test had a titre >1:64 or nephelometric DRB1*0101/2 and *0103. RA determination was >40 IU/ml on two or more HLA-DRB1*04 subtypes were distinguished by PCR occasions), age at onset of disease, presence of EAF amplification of DRB1 genes and sequence-specific and duration of the disease. All seronegative patients oligotyping using 32P-end-labelled probes, according had normal sacroiliac radiographs and a negative to the 11th Histocompatibility Workshop protocol family history of psoriasis and spondylitis. We consid- [15]. ered the following EAF: s.c. nodules, pulmonary disease (fibrosing alveolitis and pleuritis), pericarditis, Statistical analysis Felty s syndrome, Sjögren s syndrome, cutaneous vasculitis, Statistical analysis was carried out using the SPSS neuropathy, amyloidosis. The diagnostic defini- program. x2 with Yates correction and Fisher s exact tion of each EAF was reported in our previous study tests were used to compare the frequencies of HLA [4]. The healthy control group consisted of 396 blood antigens. A t-test was used when necessary. Relative donor volunteers from the same geographical area. risks ( RR) were calculated using the Woolf method. RESULTS There were no statistically significant differences when comparing the distribution of HLA-DR4 sub- types between RA patients and DR4-positive controls (Table II). A trend of a higher frequency of DR1 in RA patients compared to controls was observed ( 24% vs 16%, P = 0.08, RR 1.5). Alleles containing the rheumatoid epitope were expressed in 45% of RA patients, 51% of seropositive patients and 33% of seronegative patients. Only six patients (7%) had a double dose of rheumatoid epitope. One of the patients was homozygous for the DRB1*0401 allele, a second patient was homozygous for the DRB1*0404 allele; the other four patients carried a combination of a DR4 variant with DRB1*0101/2 alleles. Four patients were seropositive and two were seronegative. No significant differences in the presence of rheumatoid factor, EAF and articular damage were observed between patients with no, one or two doses of epitope (data not shown). TABLE II Frequency of DRB1 alleles in DR4+ RA patients compared with DR4+ healthy controls Controls Patients DR4 subtypes (n = 41) (n = 29) P RR (CI) DRB1* % 34% NS 2.0 ( ) DRB1* % 7% NS 0.5 ( ) DRB1* % 10% NS 1.1 ( ) DRB1* % 21% NS 0.9 ( ) DRB1* % 21% NS 1.2 ( ) DRB1*0406 2% 0% NS DRB1* % 7% NS 0.7 ( ) DRB1*0408 5% 0% NS DRB1*04 (epitope +)* 70% 76% NS 1.6 ( ) *HLA-DRB1*04 alleles encoding the rheumatoid epitope= B1 *0401, *0404, *0405, *0408. Radiological examination Radiographs of the hands and feet were available for all the patients, and were read separately by four of the authors (BB, TC, LM, CS) without knowledge of the identity of the patients. A blind consensus result was given when necessary. We evaluated the presence of erosions in metacarpophalangeal (MCP) joints, proximal interphalangeal ( PIP), wrists and metatarso- phalangeal (MTP) joints, and graded damage in the same joints according to the Larsen score [11], using a standard film set for comparison. For each patient, we obtained two scores: the first was the number of joints with at least one erosion (range 0 36) and the second the total Larsen score (the sum of the score of all the evaluated joints, range 0 180). HLA serological typing Serological tissue typing for HLA class II antigens was performed in patients and controls by means of a standard microcytotoxicity assay using serum sets from the 11th International Histocompatibility Workshop and some commercial antisera (One Lambda, Los Angeles, CA, USA) [12]. TABLE I Demographic and clinical data in Northern Italian patients with rheumatoid arthritis Number of patients 86 Mean age ± S.D. (yr) 60.1 ± 10.8 Mean duration of disease ± S.D. (yr) 8.7 ± 7.0 Females/males (%) 67/33 Seropositive/seronegative (%) 69/31 Mean number of eroded joints ± S.D ± 9.9 Mean Larsen score ± S.D ± 31.4 Extra-articular features (%) 51

3 SALVARANI ET AL.: HLA-DRB1 IN ITALIAN PATIENTS WITH RA 167 TABLE III higher Larsen score compared to those without the Rheumatoid arthritis features according to allelic variants of DRB1 epitope (P < 0.05). The frequency of EAF was also DRB1 alleles coding rheumatoid epitope greater, but the difference was not significant. No differences were present between patients expressing Patients with Patients with All sequence HLA-DRB1*01 alleles and those lacking the rheumat- Characteristics DRB1*04,X DRB1*01,Y& negative oid epitope. The disease duration was similar in the Patients (n) three groups of patients. Age (yr) 58.3 ± ± ± 12.0 Female (%) DISCUSSION Disease duration Recently, studies from the Mayo [ 3, 16] have clearly (yr) 7.6 ± ± ± 6.7 shown that in Caucasian RA patients of Northern Seropositivity (%) Number of eroded European ancestry the severity of the disease is strictly joints 14.4 ± ± ± 9.9* correlated to the dose of rheumatoid epitope. Patients Larsen score 65.9 ± ± ± 32.4* with a disease-linked HLA-DRB1 allele on both haplo- Extra-articular types have a more aggressive disease with extradisease (%) articular manifestations and increased frequency of X denotes any DRB1 allele other than *01. joint surgery. In particular, HLA-DRB1*0401 homo- &Y denotes any DRB1 allele other than *04. zygosity was associated with rheumatoid vasculitis. *P < 0.05 compared to DRB1*04,X. In Italy, as in other Mediterranean countries [7, 8], DR4 is weakly associated with RA [4], and in these The sequence cassette QKRAA was detected in 13% countries RA seems to be milder than in northern of seropositive and 7% of seronegative patients, while Europe with a lower frequency of extra-articular manifestations QRRAA was detected in 34% of seropositive and 26% [4 6]. In a previous study [4] from our of seronegative patients. These differences were not group, only 32% of the patients had EAF. In Italian significant. Four patients expressed the QRRAA motif RA patients, as in Greek patients, Felty syndrome and on both haplotypes, three were seropositive and one vasculitis are very rare. The higher presence of patients seronegative. Only one patient expressed two copies of with EAF in this study compared to our previous QKRAA and he was seropositive. Another one study was probably correlated to the selection of expressing QKRAA on one haplotype and QRRAA DR4-positive patients who more frequently had EAF. on the other was seronegative. Although we selected DR4-positive patients for our No significant differences in the Larsen score, study, the rheumatoid epitope was expressed by only number of eroded joints and in the frequency of extra- 45% of the patients with RA. We found the articular disease were observed in seropositive patients RA-implicated sequence in 51% of seropositive patients with either double, single or no doses of epitopes. No and in 33% of seronegative patients, unlike Weyand significant differences were observed in seronegative et al. [17], who showed the rheumatoid epitope in 97% patients as well (data not shown). Seropositive patients of seropositive patients and 68% of seronegative with no dose of epitope had a significantly higher patients. A similar low frequency of rheumatoid epi- frequency of EAF (72 vs 28%, P = 0.003) and tope ( 37.9%) has recently been reported by McDaniel a significantly higher number of eroded joints et al. [18] in African-American RA patients. As in (12.4 ± 10.4 vs 3.8 ± 6.5, P = 0.003) and a signific- African-American patients, the presence and the dose antly higher Larsen score (60.8 ± 33.8 vs 33.1 ± 21.6, of HLA-DRB1 alleles did not, in Northern Italian P = 0.002) compared to those who were seronegative. patients, correlate with the disease severity, as evaluated The disease duration was not significantly different in by the presence of erosions and EAF. This absence these two groups [11.2 ± 7.2 yr vs 6.9 ± 5.0 yr, P=not of association between the presence and the dose of significant (NS)]. Comparing seropositive and seronegative rheumatoid epitope and disease severity was observed patients with a single dose of epitope, we observed in both the seropositive and seronegative patients. no differences in either the number of eroded joints Our data do not exclude the possibility that in (13.7 ± 10.3 vs 7.1 ± 9.5, P = NS) or in the Larsen Northern Italian patients, as well, HLA MHC genes score (62.5 ± 32.4 vs 52.9 ± 23.8, P = NS), while a outside the DRB1 locus may be associated with disease significantly higher frequency of EAF was present in severity. In British patients, some EAF (in particular seropositive patients (50 vs 14%, P < 0.05). The small Felty s syndrome) are preferentially associated at the number of patients did not allow any comparisons DQB locus with the DQw7 allele and within class III between seronegative and seropositive patients with a with the C4B null allele [19 21]. These associations double dose of epitope. seem to represent a marker of this particular EAF We compared the 18 patients encoding the epitope more than a marker of articular disease severity [20, by an HLA-DR4 allele, the 17 encoding the epitope 21]. by a non-dr4 allele and the 47 patients with no dose Another similarity between African-American and of the epitope ( Table III). Four patients positive for Northern Italian patients was the very low frequency both DR4 and DR1 were excluded from the analysis. The patients encoding the epitope by an HLA-DR4 allele had a higher number of eroded joints and a of patients with a double dose of the rheumatoid epitope. Only six patients had a double dose of HLA- DRB1 alleles encoding the epitope, and only two of

4 168 BRITISH JOURNAL OF RHEUMATOLOGY VOL. 37 NO. 2 these patients were homozygous for HLA-DRB1*0401 seropositive and seronegative RA patients have a very and DRB1*0404. Two of the other four patients were low frequency of the QKRAA cassette; QRRAA was DRB1*0404/0101, one was DRB1*0401/0101 and the more represented but equally distributed between seropositive fourth DRB1*0405/0101. Therefore, we could not and seronegative patients. The higher frefourth evaluate the influence of HLA-DRB1*0401 or *0404 quency of the QRRAA cassette was connected with homozygosity in Italian RA patients because of the the high frequency of DR1-positive patients. Very few rarity of these allelic combinations. A similar rarity patients expressed two copies of these disease-linked of homozygosity for the DR4 specificity was also sequences on both haplotypes, precluding any compar- observed in Southern France (Marseille) and Spain isons with rheumatoid factor status. (Seville) [6, 8]. A world map of gene homozygosity Our study confirms the relationship between rheumwas generated by Cavalli-Sforza et al. [22]. The lowest atoid factor positivity and a more aggressive disease, values of homozygosity (and consequently the highest regardless of the presence of rheumatoid epitope. heterozygosity) are in the Middle East and Southern The seropositive patients with no dose of epitope Europe. These areas were regions of major population presented a significantly higher frequency of EAF and movements with confluence of diverse human popu- a more erosive disease compared to those who were lations. Northern Europe and the UK have higher seronegative. The association between seropositivity values of homozygosity compared to Mediterranean and EAF was also present in the patients with a single countries. The rare expression of HLA-DRB1*04 dose of epitope. The number of patients with a double homozygosity in Mediterranean patients may in part dose of epitope was too small to permit any explain the reduced predisposition for developing a observations. more aggressive disease in these populations. We did not find any significant association between It has been shown that DR1 is associated with RA seropositivity and the rheumatoid epitope. Recently, in the Italian population [23] as in other Mediterranean Nelson et al. [26] showed a correlation in women countries [6, 8]. Recently Angelini et al. [24] found a between seropositivity and DR4, but not with DR1. trend of a positive association with DRB1*0101 in 27 Seropositivity was not significantly increased in our DR4-negative, Italian RA patients. We, too, observed DR4-positive patients compared to DR1-positive a trend of a higher frequency of DR1 subtypes in our patients. However, a trend of increased frequency of patients compared to controls. rheumatoid factor in DR4-positive patients vs patients The DR4 patient selection and the high frequency without DR4 and DR1 was observed. of DR1 patients permitted us to compare patients Nelson et al. [ 26] observed a decline of DR4 positiv- encoding the epitope by an HLA-DR4 allele with those ity in older seropositive RA patients. The proportion encoding it by a non-dr4 epitope. Interestingly, only of patients who were elderly at the onset of the disease the patients encoding the epitope by an HLA-DR4 was higher in our study compared to that of Nelson allele had more erosive disease; the frequency of extraarticular et al. (76% of our seropositive patients had RA onset manifestations was also higher in this sub- at age >53 yr, while only 26% of those in the Nelson group of patients, even if the difference was not significant. et al. study had RA onset at age >53 yr). Therefore, The QRRAA cassette contained in DRB1*0101 more patients who were rheumatoid factor positive and *0102 alleles seems not to be correlated with a but DR4 negative were probably present in our study. more destructive disease. Our data confirm the study In conclusion, our data suggest that in the Northern of Olsen et al. [25]. These authors also observed a Italian population the radiological severity of disease significant association between DR4 and more erosive is connected to HLA-DRB1*04 subtypes. HLA- RA, while DR1 was not associated with more severe DRB1*01 alleles seem not to influence disease severity. radiographic changes. Rheumatoid factor positivity is correlated to disease Our data confirm the thesis of Weyand et al. [17], severity even in the absence of any rheumatoid epitope supporting the key role of HLA-DRB1*04 subtypes in association. 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