A new classification of HLA-DRB1 alleles differentiates predisposing and protective alleles for autoantibody production in rheumatoid arthritis

Since early rheumatoid arthritis (RA) is often indistinguishable from other inflammatory joint diseases, recent-onset inflammatory synovitis poses a diagnostic and prognostic challenge to rheumatologists [1]. The identification and validation of immunologic and genetic markers with strong diagnostic and prognostic value in early RA may help rheumatologists to meet this challenge [2].

Among immunologic markers, anticitrullinated protein/peptide antibodies (ACPAs) constitute relevant tools in the diagnosis and prognosis of early RA. Citrulline is a nonstandard amino acid, generated by post-translational modifications of several proteins by deimination of arginine residues by peptidylarginine deiminases [3, 4]. The citrulline moiety is the true determinant in proteins recognized by antiperinuclear factor [5], antikeratin antibodies [6], antifilaggrin antibodies [7], anticyclic citrullinated peptide (anti-CCP) antibodies [8] and anti-deiminated human fibrinogen autoantibodies (AhFibA) [9‐11]. ACPAs may be detected in healthy individuals, years before the onset of symptoms of RA [12, 13], and may predict progression to persistent erosive arthritis or to RA in patients with undifferentiated arthritis [14‐16]. ACPAs are as sensitive as, and more specific than, rheumatoid factor (RF) for early RA diagnosis [17‐19]. Furthermore, ACPAs represent a prognostic factor for erosive disease in early RA [20‐24].

Among genetic markers, the HLA-DRB1 gene has been clearly involved in the pathogenesis of RA [25, 26]. The association between HLA-Dw4 and RA was first reported in 1976 [27]. The development of HLA-DRB1 genotyping led to the demonstration that different HLA-DR4 alleles were not equally associated with RA and that several non-DR4 HLA–DRB1 alleles were also associated with the disease. The shared epitope (SE) hypothesis, first proposed in 1987, represents an approach to understand the molecular genetics of susceptibility to RA. The SE hypothesis assumes that HLA-DRB1 alleles encoding a highly conserved amino acid sequence, known as the SE – which is characterized by the RAA pattern at positions 72–74 of the third hypervariable region of different HLA-DRβ₁ chains – are associated with susceptibility to RA [28]. HLA-DRB1 alleles encoding the SE were then associated with structural severity of RA [29] and have been more recently associated with production of ACPAs [9, 12, 24, 30‐32].

As was done in previous attempts [33, 34], du Montcel and colleagues recently introduced a new classification of HLA-DRB1 alleles that reconsiders the SE hypothesis [35]. In terms of susceptibility to RA, this new classification suggests that the risk of developing RA depends on whether the RAA sequence occupies positions 72–74 but the risk is modulated by the amino acids at position 71 (K confers the higher risk, R an intermediate risk, A and E a lower risk) and at position 70 (Q or R confers a higher risk than D) [35‐37] complexifying the classical SE epitope classification based on the presence of RAA in positions 72–74. In terms of structural severity of RA, this new classification allowed the differentiation of predisposing or protective alleles (two effects) – respectively characterized by the DRRAA or by the DERAA amino acid pattern at positions 70–74 [36] – which was not possible using the classical SE epitope classification based on the only presence of RAA in positions 72–74.

In the present study, we investigated the relevance of this new classification of HLA-DRB1 alleles in terms of RF and ACPA production in a cohort of French Caucasian patients with early RA. Interestingly, the new classification of HLA-DRB1 alleles allows the differentiation between predisposing and protective alleles for autoantibody production.

The results of the present study confirm previous evidence of an association between HLA-DRB1 gene polymorphisms and RF or ACPAs in RA. Furthermore, the results point out the interest of the new classification of HLA-DRB1 alleles in order to differentiate predisposing and protective alleles for autoantibody production in early RA.

The results of the present study confirm previous evidence of an association between HLA-DRB1 gene polymorphisms and autoantibody production in RA. We found a positive association between carriers of HLA-DRB1*SE⁺ alleles (HLA-DRB1*0401, HLA-DRB1*0404, HLA-DRB1*0405, HLA-DRB1*0408, HLA-DRB1*1001) and RF or ACPA production, while we did not find any negative association between carriers of HLADRB1*SE^- alleles and RF or ACPA production (data not shown). An association between HLA-DRB1*04 or HLA-DRB1*SE⁺ alleles and RF has been reported in some studies [12, 30, 40] but rejected in others [12, 32, 41]. An association between HLA-DRB1*01, HLA-DRB1*04 or HLA-DRB1*SE⁺ alleles and ACPAs was more constantly reported in European or North American RA patients [9, 12, 24, 30, 32, 40, 42‐45]. Since the presence of RF was strongly correlated with that of ACPAs in most of these studies, several groups investigated whether these associations between HLA-DRB1 gene polymorphisms and RF or ACPAs were independent. These studies showed that the association between HLA-DRB1*SE⁺ alleles and ACPAs is constantly stronger than the association between HLA-DRB1*SE⁺ alleles and RF. Furthermore, they suggested that the association between HLA-DRB1*SE⁺ alleles and ACPAs is independent of the RF status, leading to the conclusion that HLA-DRB1*SE⁺ alleles are primarily associated with the presence of ACPAs, but not with the presence of RF [24, 32, 41].

The results of the present study indicate the interest of the new classification of HLA-DRB1 alleles to differentiate predisposing and protective alleles for autoantibody production in early RA. This new classification, which is based on an initial split of HLA-DRB1 alleles into two groups according to the presence (S alleles) or absence (X alleles) of the RAA sequence at positions 72–74, subsequently divides S alleles into four groups according to the amino acids at positions 71 and 70. Most of the previous studies, based on the common classification, identified HLA-DRB1*101, HLA-DRB1*0401, HLA-DRB1*404 and HLA-DRB1*1001 as predisposing alleles for ACPA production in RA, with a significant dose effect in patients carrying two of these predisposing alleles [9, 12, 32, 44]. Only a few association studies reported an HLA-DRB1 allelic protective effect for ACPA production in RA. In these studies, HLA-DRB1*03 was associated with ACPA-negative RA and decreased titers of ACPAs, even in the presence of an SE allele [32, 45]. In the new classification of HLA-DB1 allelles, HLA-DRB1*03 is not taken into account separately since it is classified into the X group of alleles, which do not encode the SE sequence. In the present study, complementary analysis did not show any association between HLA-DRB1*03 carrier status and RF or ACPA production (data not shown). The use of the classification by du Montcel and colleagues suggests a risk hierarchy in ACPA production in early RA patients: the S2 (KRAA at positions 71–74) and S3P (QRRAA or RRRAA at positions 70–74) alleles conferring predisposition, while the S1 (ARAA or ERAA at positions 71–74) and S3D (DRRAA at positions 70–74) alleles confer protection, in comparison with X (non-RAA patterns at positions 72–74).

The use of the new classification of HLA-DRB1 alleles proposed by du Montcel and colleagues seems to provide different pictures of the relative contribution of the HLA-DRB1 locus to RA pathogenesis. This relative contribution is not restricted to ACPA production, but also includes risk hierarchy for RA susceptibility and structural severity [35‐37].

Trying to understand the findings of genetic association/linkage studies in complex multifactorial diseases, such as RA, in light of the amino acid alignment of a protein encoded by a candidate gene remains a challenging task. In fact, the interactions between HLA-DRB1 molecules and citrullinated peptides may impact RA pathogenesis in several ways. For example, a previous study conducted in DR4-IE transgenic mice demonstrated that the conversion of arginine to citrulline at the peptide side-chain position interacting with the SE significantly increases peptide–MHC affinity and leads to the activation of CD4⁺ T cells, suggesting that HLA-DRB1 alleles encoding the SE could initiate an autoimmune response to citrullinated self-antigens [46].

Although no formal conclusions on causality can be drawn from the present association study, our findings indicate the interest of this new classification of HLA-DRB1 alleles in order to differentiate predisposing and protective alleles for autoantibody production in RA.