Arthritis Research Campaign Epidemiology Unit, School of Epidemiology and Health Sciences, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT
1 Centre for Paediatric and Adolescent Rheumatology, UCMLS, London W1P 6DB and
2 Department of Rheumatology, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
Abstract
Objective. To determine the HLA associations with juvenile idiopathic arthritis (JIA) and its subgroups as defined by the International League of Associations for Rheumatology (ILAR) classification criteria.
Methods. Five hundred and twenty-one UK Caucasian JIA patients and 537 UK Caucasian controls were typed for HLA class II alleles. Phenotype and haplotype frequencies were compared between all JIA cases and controls and between the seven ILAR-defined JIA subgroups.
Results. Three haplotypes (DRB1*08-DQA1*0401-DQB1*0402; DRB1*11-DQA1*05-DQB1*03; DRB1*1301-DQA1*01-DQB1*06) were associated with increased risk and one (DRB1*04-DQA1*03-DQB1*03) with decreased risk of JIA. However, in each case the frequencies also varied between JIA subgroups.
Conclusion. This study categorically demonstrates that there are multiple HLA class II associations with JIA. It has also, for the first time, defined these associations in the seven different ILAR subgroups in UK JIA cases. Although there are a number of common associations, each ILAR subgroup exhibits different patterns of HLA associations, suggesting that the ILAR classification system does define genetically distinct groups of patients.
KEY WORDS: JIA, HLA.
Juvenile idiopathic arthritis (JIA) is a clinically heterogeneous group of diseases, the study of which has been hindered by the lack of a classification system that is accepted internationally. Juvenile arthritis was initially classified into three subgroups (pauciarticular, polyarticular and systemic-onset disease) on the basis of the number of joints affected at disease onset and a variety of extra-articular clinical features (ARA [1], European League Against Rheumatism (EULAR) [2]). Additional classifications were proposed for spondyloarthropathy [3] and psoriatic arthritis [4, 5]. Recently, a working group of The International League of Associations for Rheumatology (ILAR) proposed [6] and modified [7] a new unified classification system to minimize international differences in disease definition and to identify clinically homogeneous disease subgroups within the umbrella term JIA. This classification system included seven JIA subtypes as well as an unclassifiable group. All JIA subtypes are characterized by chronic articular inflammation and, in common with other chronic inflammatory conditions, extensive and well-documented associations with HLA alleles have been described [8]. The earliest report was of an association between HLA-B27 and older children with pauciarticular onset disease [9]. Other associations have been noted, the most studied being early-onset pauciarticular disease, where there have been numerous reports of associations with HLA-A2, DRB1*08 and *11 and DPB1*0201 (reviewed in [10]).
The difficulty in interpreting HLA associations in JIA is two-fold. First, many of the previous studies have been based on relatively small numbers, which makes the results of studying associations with a highly polymorphic genetic system such as HLA difficult to interpret. Secondly, the lack of a universally agreed classification system makes comparison between studies virtually impossible. The question we wanted to address is whether the clinically homogeneous disease subgroups defined by ILAR were also genetically distinct from each other. Strong associations with HLA that either confirm previously reported associations or represent previously unrecognized associations would support this. Our hypothesis was that some HLA class II alleles or defined haplotypes represented predisposing risk factors for all types of JIA, whereas others are associated with specific subgroups of JIA only.
Materials and methods
Patients
Five hundred and twenty-one UK Caucasian patients from 17 centres within the UK were recruited through the British Paediatric Rheumatology Group (BPRG). Patients were classified into one of the seven ILAR subgroups by the clinicians according to the revised ILAR classification criteria [7]. A 12 ml sample of EDTA blood was provided for DNA extraction.
Controls
Five hundred and thirty-seven UK Caucasian controls were available for comparison. These originated from three sources: 118 were from general practice registers as comparative subjects for the Norfolk Arthritis Register (NOAR) [11], the second group comprised 159 individuals from the same region of England, collected as part of a population-based survey identifying possible risk factors for cancer [12], and the third group was a cohort of 260 UK blood donors collected as controls for disease studies.
HLA typing
HLA-DRB1, DQB1 and DPB1 alleles were determined in all patients using a commercially available semi-automated PCR-SSOP (sequence specific oligonucleotide probes) typing system (Inno-LiPA; Abbotts, Maidenhead, UK). HLA-DQA1 alleles were determined using a PCR-SSOP system [13]. In addition, the majority of patients were also typed for HLA-B27 using PCR-SSP (sequence specific primers). All 537 controls were typed for HLA-DRB1, 137 for DQA1, 155 for DQB1 and 128 for DPB1. As strong linkage disequilibrium exists across the MHC class II region, DRB1-DQA1-DQB1 haplotypes were assigned in individuals where data for all three loci were available. These haplotypes were assigned on the basis of well-documented known haplotypes for Caucasians [14]. Three-locus haplotypes have been assigned for 346 patients and 121 controls.
Statistical analyses
The phenotype frequency (i.e. frequency of possessing at least one copy) of each HLA allele and each haplotype was calculated for controls, for all JIA cases and for each subtype of JIA patients. Phenotype frequencies were compared between all JIA cases and controls using the 2 test and associations were expressed as odds ratios (ORs) with their 95% confidence intervals (CIs).
The main focus of this study was to investigate HLA associations with subtypes of JIA defined by the ILAR classification. Comparing each of the ILAR subtypes separately with controls for each of the HLA phenotypes of interest would result in a very large number of hypothesis tests. We therefore adopted the strategy of first examining whether there was evidence of a difference in the presence or absence of each phenotype between the seven ILAR subgroups. Differences between subgroups were assessed using 2 tests on the 7 x 2 tables; if numbers in some categories were small, Monte Carlo simulation was used to estimate exact P values. No adjustment was made for multiple testing. Only where evidence for a difference was found (at a liberal 10% significance level) were ORs calculated comparing each JIA subgroup with controls. With this strategy there is a greater chance of failing to detect some subgroup associations but the multiple testing problem is reduced.
Confidence intervals were calculated using the Cornfield approximation [15]. All analyses were carried out in Stata (Stata Corporation, TX, USA) [16] except for the Monte Carlo methods, where SPSS 8.0 for Windows (SPSS Inc., Chicago, IL, USA) was used.
Results
Five hundred and twenty-one Caucasian JIA patients and 537 controls were available for study. The numbers of patients, along with median age at onset, male/female ratio and frequency of HLA-B27 for each ILAR subgroup, are given in Table 1. There were no differences in the phenotype frequencies between each of the three control groups and so they were pooled for further statistical analysis.
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In all cases where a difference was seen between all JIA cases and controls there was also a difference between the seven JIA subgroups. In addition, some alleles/haplotypes that did not differ between all JIA cases and controls did show a difference between subgroups.
Subgroup associations
Two HLA-DRB1 alleles were associated with increased risk (DRB1*08 and 11) and two with decreased risk (DRB1*04 and *07) of JIA. These phenotype frequencies also differed between subgroups (Table 3a).
The increased risk associated with HLA-DRB1*08 was largely attributable to persistent oligoarthritis, extended oligoarthritis and RF-negative polyarthritis, with odds ratios of 3.9, 6.3 and 3.1 respectively. For DRB1*11 the increase was seen in systemic arthritis (OR 2.8) as well as the persistent (OR 2.5) and extended oligoarthritis (OR 2.5) subgroups. HLA-DRB1*04, which is known to be associated with adult RA, was only associated with an increased risk for RF-positive polyarthritis (OR 3.2, 95% CI 1.66.5). In all other subgroups except systemic arthritis and enthesitis-related arthritis (ERA) there was decreased risk associated with HLA-DRB1*04. The reduced risk of JIA associated with the HLA-DRB1*07 allele was strongest for persistent oligoarthritis, RF-positive and -negative polyarthritis and ERA.
Some alleles at the HLA-DQA1 locus were associated with increased risk (DQA1*0103, *04 and *05) and others decreased risk (DQA1*02 and *03) of JIA (Table 3a). The risk associated with HLA-DQA1*0103 was most pronounced in persistent oligoarthritis. DQA1*04, which is in linkage disequilibrium with DRB1*08, was increased in persistent and extended oligoarthritis as well as RF-negative polyarthritis. HLA-DQA1*05 was also associated with persistent oligoarthritis (OR 2.6) as well as systemic arthritis (OR 2.5).
HLA-DQA1*03, which is in linkage disequilibrium with HLA-DRB1*04, was most reduced in persistent oligoarthritis, extended oligoarthritis and psoriatic arthritis, and was increased in RF-positive polyarthritis. HLA-DQA1*02 was particularly reduced in persistent oligoarthritis and RF-positive polyarthritis.
Only one allele at the HLA-DQB1 locus, DQB1*04, was associated with JIA as a group, conferring an increased risk (OR 3.5) (Table 3a). This was most pronounced in persistent oligoarthritis and extended oligoarthritis. This is in keeping with the results at other loci, as HLA-DQB1*04 is in linkage disequilibrium with HLA-DRB1*08 and DQA1*04.
Two further HLA-DRB1 alleles (*01 and *13) differed between JIA subgroups, although there was no difference between JIA as a whole and controls. Possession of an HLA-DRB1*01 allele was associated with an increased risk of ERA and psoriatic arthritis (OR 3.6 and 2.7 respectively compared with controls). In the ERA patients there was a high frequency of HLA-B27 (75%), which is in linkage disequilibrium with HLA-DRB1*01. However, the increase in HLA-DRB1*01 appeared to be independent of HLA-B27 status (Table 4). HLA-DQA1*0101 was also increased in subjects with ERA and psoriatic arthritis (OR 2.8 and 4.2 respectively). This allele is in linkage disequilibrium with HLA-DRB1*01. HLA-DRB1*13 was only found to be increased in persistent oligoarthritis.
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Associations by haplotype
The associations seen at individual loci are reflected in the haplotypic associations seen. Three haplotypes (DRB1*08-DQA1*0401-DQB1*0402; DRB1*11-DQA1*05-DQB1*03; DRB1*1301-DQA1*01-DQB1*06) were associated with increased risk and one (DRB1*04-DQA1*03-DQB1*03) with decreased risk of JIA as a whole (Table 3b). The risk associated with the DRB1*08-DQA1*0401-DQB1*0402 haplotype was largely attributable to persistent oligoarthritis and extended oligoarthritis, with odds ratios of 6.1 and 10.3 respectively. The DRB1*11-DQA1*05-DQB1*03 haplotype was also increased in persistent and extended oligoarthritis as well as in systemic arthritis.
The decreased risk associated with the haplotype DRB1*04-DQA1*03-DQB1*03 was most pronounced in persistent and extended oligoarthritis; as expected, the risk of RF-positive polyarthritis was greater for subjects with this haplotype (OR 3.9).
Two further haplotypes (DRB1*01-DQA1*0101-DQB1*0501; DRB1*0701-DQA1*02-DQB1*02) differed between JIA subgroups. The haplotype DRB1*01-DQA1*0101-DQB1*0501 was associated with an increased risk of ERA and psoriatic arthritis (OR 4.9 and 3.8 respectively compared with controls), whereas the haplotype DRB1*0701-DQA1*02-DQB1*02 was associated with a decreased risk of persistent oligoarthritis JIA (OR 0.2).
Discussion
This is one of the largest HLA association studies with JIA to date and the first to present HLA class II phenotype and haplotype frequencies in each of the seven ILAR subgroups of JIA. We have confirmed that multiple HLA class II associations exist with JIA. We have shown that each ILAR subgroup has a characteristic pattem of HLA associations, suggesting that the ILAR classification system does define genetically distinct groups of patients. However, there are also many common associations between some of the groups, particularly oligoarthritis, extended oligoarthritis and RF-negative polyarticular JIA (which may indicate that the ILAR classification system could be further modified).
There have been many previous reports of HLA associations in JIA. However, these studies have mostly been based on relatively small numbers of patients and have used a variety of different classification systems. It is difficult, therefore, to establish whether the differences seen in these studies represent a true reflection of genetic heterogeneity in JIA or are merely a reflection of the clinical heterogeneity in the patients studied. Even the most recent data collected for the 12th Histocompatibility Workshop, where all patients had been classified according to ILAR criteria, are confusing, as patients and controls from a variety of different ethnic backgrounds have been pooled for analysis [17]. More recently, Pratsidou-Gertsi [18] has independently analysed the 228 Greek patients submitted to the workshop. The patients have been classified using ILAR criteria but in addition have been subdivided by other factors, such as ANA status and age at onset of disease. Age at onset was deliberately left out of the ILAR classification as, although it is thought to be important, the cut-off age has always been assigned arbitrarily. Murray et al. [19] have recently used HLA data on 680 JIA patients to try to define the cut-off for age at onset. They suggest that HLA-specific windows of susceptibility unique to each subtype of JIA exist. These findings need to be confirmed in another large data set, such as the one described here, and this work is currently under way.
The large numbers of clinically well-defined patients and controls in our study represent a relatively homogeneous sample from an ethnic perspective and ensure more accurate estimates of risk and narrower confidence intervals than in previous studies. The association we see with the haplotypes DRB1*08-DQA1*04-DQB1*04 and DRB1*11-DQA1*05-DQB1*03 in persistent and extended oligoarthritis are consistent with previous studies [20]. HLA-DRB1*13 was also increased in oligoarthritis. The increase in HLA-DRB1*13 in this subgroup may reflect the high frequency of ANA-positive patients in this ILAR subgroup. We have reported previously an association between HLA-DRB1*1301 and ANA positivity in JIA [21]. More recently, a study in which we used the statistical approach of latent class analysis to identify subgroups in JIA also showed HLA-DRB1*13 to be a significant risk factor in subgroups where patients were ANA-positive but not in ANA-negative subgroups [22]. HLA-DRB1*01 is associated with ERA and psoriatic arthritis and this association is independent of HLA-B27. This may suggest a role for additional gene(s) within the MHC in these conditions. Consistent with many previous studies [23] is the decrease in HLA-DRB1*04, an allele which is strongly associated with adult RA. It is difficult to establish whether or not this is a real effect; obviously if a number of alleles are raised in frequency then others must be reduced.
There are some challenges in conducting genetic association studies, even in a population of this size. Although it is very useful to summarize these findings in a single report, comparison between seven subgroups of disease for four highly polymorphic loci inevitably leads to problems of multiple testing. Our statistical strategy significantly reduces this problem, although it may fail to detect some subgroup associations.
Another problem is that the ILAR classification is not uniformly based on an end-point diagnosis but diagnosis at a minimum disease duration of 1 yr. However, the disease duration for patients in this study extended up to 16 yr so that some of the patients may well have reached their final diagnoses and others may not. This is more critical for some of the subgroups: for example, it is very difficult to define persistent oligoarthritis, as many of the patients originally diagnosed as having oligoarthritis may later develop more joint involvement and therefore be reclassified as having extended oligoarthritis. Conversely, patients classified as having RF-negative polyarthritis may well have started out with a diagnosis of oligoarthritis JIA. This may well account for some of the common genetic associations between the groups. It may be that a time point diagnosis may be more appropriate in JIA, each patient being classified at yearly intervals following disease onset.
It would be very interesting to compare the ILAR classification systems with previous classification systems, such as EULAR and ARA, but in reality this is very difficult as there is no validated way of comparing the strength of associations seen. In addition, a cross-tabulation between ILAR and EULAR classifications shows that 93% of patients classified as having systemic arthritis or RF-negative or RF-positive polyarthritis fall into the same category in both classifications and patients now classified as having psoriatic arthritis would also have been classified previously as having psoriatic arthritis by the Vancouver criteria. Therefore it is only necessary to consider patients classified by ILAR as having persistent or extended oligoarthritis, both of which were previously grouped together as pauciarticular, and those classified as having ILAR ERA who previously fell into a variety of different categories. As far as HLA is concerned, there are no striking differences between persistent and extended oligoarthritis patients, with the possible exception of the DRB1*13-DQA1*01-DQB1*06 haplotype, which is only increased in persistent oligoarthritis disease. This does not rule out the possibility that there are other genetic differences between these two groups. The ERA association with the haplotype DRB1*01-DQA1*0101-DQB1*0501 suggests that this is a genetically distinct group of patients.
It is important when establishing a new classification system for any disease to ensure that it is biologically relevant. It is also important to define subgroups practically, so that patients who need to be managed/treated similarly are grouped together. From the point of view of understanding the genetics of a complex condition such as JIA, it is essential to define genetically homogeneous groups of patients. HLA associations identified in JIA may help to define more homogeneous groups of patients, but identifying other genetic risk factors may lead to better definition of JIA subgroups and hence clarification of some of the HLA associations. To be able to progress in our understanding of the genetics of JIA, future studies need to be undertaken on large cohorts of patients, from defined ethnic backgrounds, classified according to the same criteria. Accurate outcome data are also essential if we are to achieve our ultimate aims of being able to predict outcome and establish new therapies for children with JIA.
Acknowledgments
This work was supported by the Arthritis Research Campaign.
Notes
Correspondence to: W. Thomson, Arthritis Research Campaign Epidemiology Unit, School of Epidemiology and Health Sciences, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK.
Contributors to the British Paediatric Rheumatology Study Group: A. Bell, Belfast; A. Craft, Newcastle; J. David, Battle, Reading; J. Griffin, Enfield; A. Hall, Wrexham Park, Slough; M. Hall, Cardiff; A. Herrick, Hope Hospital, Manchester; P. Hollingworth, Bristol; L. Holt, ARC, Manchester; S. Jones, Blackpool; G. Pountain, Huntingdon; C. Ryder, Nuneaton; T. Southwood, Birmingham; I. Stewart, Blackpool; P. Woo, Great Ormond Street and Middlesex Hospitals; S. Wyatt, Leeds; H. Venning, Nottingham.
References