Introduction

The pathogenesis of rheumatoid arthritis (RA) is multifactorial, involving both genetic and environmental factors. Although associations between some HLA-DRB1 alleles and RA were reported nearly three decades ago, the biological mechanism underlying this association remains unknown. The presence of the RAA sequence at positions 72–74 of the HLA-DR β-chain molecule for all HLA-DRB1 alleles known to be associated with RA led to the shared epitope (SE) hypothesis [1]. This hypothesis received support from numerous case-control association studies in both Caucasian and non-Caucasian populations. However, studies testing the SE hypothesis have rejected this simple model, which stipulates that each SE allele confers the same risk [2–5].

Recently, Tezenas du Montcel and coworkers proposed a model of the SE component in RA [6]. Those investigators reconsidered the SE hypothesis and generated a new classification of HLA-DRB1 alleles, based on their investigation using the MASC (Marker Association Segregation Chi Square) method [7], which was conducted in 100 trio families (one case and both parents) and 132 index cases from affected sibling pair families, all from the French Caucasian population. They proposed that the risk for develo** RA depends on whether the RAA sequence occupies positions 72–74 and, if this is the case, on the amino acids at positions 71 and 70. For those RAA alleles, lysine (K) at position 71 conferred the highest risk, arginine (R) an intermediate risk, and alanine (A) or glutamic acid (E) the lowest risk. Glutamine (Q) or arginine (R) at position 70 conferred greater risk than did aspartic acid (D). This resulted initially in five allele groups, which were simplified to three allele groups defining six genotypes with different RA risks. This study was the first to model the HLA component in RA taking into account both association and linkage data, resulting in a reshaped SE hypothesis.

Here, we tested this classification for validity by replication in a new, independent sample of 100 French Caucasian trio families, evaluating the risk hierarchy of the proposed classification for homogeneity with that of the initial sample.

Materials and methods

Study design and study population

An association study using conditional logistic regression was performed to investigate the hierarchy of risks associated with HLA-DRB1 genotypes in an independent sample of trio families. The new independent sample (sample B), similar to that used to generate the new classification (sample A), included 100 trio families (one RA patient and both parents) of French Caucasian origin (criteria fulfilled for each of the four grandparents). DNA from all of the trio families included in samples A and B was collected between 1994 and 1998, as were initial clinical characteristics of the RA index patients. RA diagnosis met the 1987 American College of Rheumatology (formerly, the American Rheumatism Association) criteria [8]. All individuals provided written informed consent, and the study was approved by the Hospital Bicêtre ethics committee (Kremlin-Bicêtre, Assistance Publique-Hôpitaux de Paris).

Clinical characteristics were updated in 2001 and 2002 for sample A and in 2004 for sample B. Four RA index patients in sample A and two RA index patients in sample B died between the time of DNA collection and the present study. The updated clinical characteristics of sample B were similar to those of sample A (the initial sample): 90% of RA patients in sample B were female versus 87% in sample A; the mean (± standard error) age at RA onset was 31 ± 9 years versus 32 ± 10 years; the mean (± standard error) disease duration was 16 ± 8 years versus 18 ± 7 years; erosions were present in 79% versus 90%; 76% were positive for serum rheumatoid factor versus 81%; and nodules were present in 19% versus 31%. Rheumatoid factor was considered positive when there was at least one positive rheumatoid factor finding during the course of the disease, as determined using latex fixation, Waaler Rose assay, or laser nephelometry.

HLA-DRB1 genoty**

Blood samples were collected for DNA extraction and genoty**. HLA-DRB1 ty** was performed using the polymerase chain reaction-sequence specific primer (SSP) method using Dynal Classic SSP DR low resolution and the Dynal Classic high resolution SSP (Dynal Biotech, Lake Success, NY, USA) for subty** of HLA-DRB1*01, *04, *11, *13 and *15 alleles. Sequencing of exon 2 of HLA-DRB1 was performed for all four HLA-DRB1*04 alleles, ambiguous with the Dynal Classic method. HLA-DRB1 allele frequencies of control genotypes (obtained by combining untransmitted parental alleles for each family) were similar between samples and were comparable to the allele frequencies reported for the French population in the 11th Histocompatibility Workshop [9].

HLA-DRB1 allele classification

HLA-DRB1 alleles were divided in two groups according to the presence or absence of the RAA sequence at positions 72–74, defining S and X alleles (Table 1). The S alleles were then subdivided into three categories, according to amino acid at position 71, as follows: S1 when an alanine or a glutamic acid was present at position 71 (A-RAA or E-RAA sequences; A-RAA alleles were too infrequent not to be pooled, as described previously [6]); S2 when a lysine was present (K-RAA sequence); and S3 when an arginine was present at position 71 (R-RAA sequence). Then S3 alleles were subdivided according to amino acid at position 70: S3D alleles encoding the D-R-RAA sequence and S3P alleles encoding the Q or R-R-RAA sequence. Because the S2 alleles had either Q or D at position 70, they had – by this '70-71-72/74' nomenclature – the Q or D-K-RAA sequence.

Table 1 Classification of HLA-DRB1 alleles
Full size table

Statistical analysis

We first investigated transmission of the five alleles (S1, S2, S3D, S3P and X) using a χ2 test with one degree of freedom for each allele. Alleles with significant over-transmission from heterozygous parents to RA patients (>50%) are linked to and associated with RA. Alleles with significant under-transmission (<50%) exhibit no RA association and could be pooled for further analysis.

Then, for each genotype 'I', the odds ratio 'ORi' relative to a reference genotype and 95% confidence interval (CI) were calculated by conditional logistic regression. In this analysis, the genotypes observed for the RA patients were conditioned to the parents' genotypes [10, 11]. The RA patient genotypes were compared using a likelihood ratio test with the pseudo-controls (i.e. the three other genotypes that could be formed by parental gametes). Given reference genotype with baseline risk termed β0, each OR βi (i = 1 ... n) was estimated by the maximization of the log likelihood (L):

ln(L) = β0 + β1X1 + β2X2 + ... + β n X n

Where ** 95% CIs were observed only between the S2/S3P highest risk genotype (OR 22.2, 95% CI 9.9–49.7) and the S3P/L lowest risk genotype (OR 4.4, 95% CI 2.3–8.4). A significant difference between other associated genotypes remains to be established. This would provide major clues that may help in deciphering the genetic component of RA, if significant differences could be correlated with distinct pathophysiological mechanisms. It was recently reported that the SE-RA association was confined to rheumatoid factor positive patients [13] or to anti-citrullin positive RA patients [14]. The precise relationship between the HLA risk genotypes and rheumatoid factor or anti-citrullinated peptide antibodies should therefore also be determined. The interaction between HLA-DRB1 genotypes and any new RA gene established by association and linkage, such as PTPN22 [15, 16], could be investigated taking this new classification into account. Ultimately, this could help in identifying other RA genetic factors that may specifically interact with only one of the HLA-DRB1 genotypes. Several previous studies indicated that other genes within HLA, such as the HLA class III region, probably contribute to RA risk [21]) and 86 (as proposed by Gao and coworkers [22]).

Because numerous association studies have suggested that the primary role played by the SE might lie in the development of severe RA [23], the relevance of this classification should be evaluated for RA prognosis in prospective cohorts. A first investigation with the new classification already provides some support for a correlation with progression of radiographic damage [24]. Indeed, it would be of great help to be able to identify those RA patients at risk for development of more severe disease, who may require more aggressive therapeutic management than patients with better prognosis.

Conclusion

In the present study we validated a first model of the effect of HLA-DRB1 on RA, resha** the SE hypothesis and providing initial estimates for the resulting risk genotypes. Building on this new HLA genotype classification could lead to improvement in our understanding of the genetics, pathophysiology and potential clinical use in management of RA.