**2. Genetic Predisposition for Rheumatoid Arthritis**

The mechanisms for RA development are similar to those of other autoimmune diseases—an immune response to the patient's self-proteins develops, which drives a cascade of proinflammatory signalling pathways and activates the production of corresponding cytokines and chemokines. In some cases, rheumatoid factor (RF) and anticitrullinated protein antibodies (ACPAs) can be found in patients before any clinical manifestations of RA. Proposed in 2010, the RA classification is based on the presence of ACPAs, found in 90% of patients. These antibodies are considered as more distinctive immunological markers of RA than others, while citrulline compounds seem to play an important role in the disease pathogenesis [3,4].

Unfavourable alleles of various genes have a relatively small influence on the disease risk when they appear individually, but in combination, they genetically predispose an individual to RA development. Currently, more than 100 loci associated with RA have been described [5]. These changes can also be found in genes whose products are not directly involved in immune reactions. The variants associated with RA pathogenesis can be found in a wide range of genes that mediate cell signalling pathways.

### *2.1. Role of HLA Genes in Rheumatoid Arthritis Development*

Based on data from the genomewide association study (GWAS), the main genes for RA predisposition belong to the major histocompatibility complex (MHC) locus. This locus, spanning 4 Mbp, occurs in the 6p21.3 region and contains about 250 genes coding the antigen proteins for T-cell recognition. Up to 60% of possible polymorphisms (genetic variants) responsible for RA susceptibility are considered to arise from the MHC locus [6]. For instance, HLA-DRB1 alleles encode unfavourable variants of the shared epitope (SE), which is critical for the correct T-cell antigen presentation process. Eighteen percent of the ACPA-positive and 2.4% of the ACPA-negative RA hereditary component is associated with these alleles. Predisposing HLA-DRB1 SE alleles are found in 64–70% of RA patients and 55% of their healthy relatives, while only being found in 35% of the average population [7].

Sequencing of the HLA-DRB1 alleles encoding the polymorphic β-chain of the DR molecule shows a prevalence of \*04:01 and \*04:04 alleles in RA patients in Europe and the \*04:05 allele in East Asia. Alleles \*04:02 and \*04:03 are shown to be protective. Based on these data, the hypothesis of the importance of the conserved epitope structure, which forms the third β-chain hypervariable region (positions p.70-74) for RA development, was postulated: whether it contains the unfavourable variants, i.e., QKRAA, QQRAA, KKRAA, QRRAA, RRRAA, or, on the contrary, the protective structure of DERAA. Moreover, amino acid substitutions at positions 11 and 13 of DR4 are also associated with RA (alleles \*04:01/\*04:04/\*04:05 compared to DR1 \*01:01) [1,8,9]. Association of the ACPAs and the hypervariable epitope alleles indicates the role of these variants in the T-cell presentation of citrullinated proteins and the further development of autoreactivity. In addition, some of the specific positions of amino acid residues suggest their participation in HLA-DR intracellular transport. Citrulline

residues are located inside the DRB1\*04:01/04 positively charged P4 pocket of the peptides but not in the negatively charged P4 pocket (encoded by the protective allele \*04:02), as shown by model experiments and crystal structure analysis. Namely, the P4 amino acid residues at positions 13 and 71 directly interact with the citrulline, so their alterations are believed to be the most relevant for RA development [10,11].

As noted above, polymorphisms leading to substitutions of amino acid residues at positions 11, 13, 71, and 74 of HLA-DRB1, as well as at position 9 in HLA-B and HLA-DPB1, have the greatest association with RA development. These residues are faced towards the protein pocket to bind the antigen in HLA molecules. These relations are more evident in African Americans and Asians compared to Caucasians. In contrast, HLA-DR3 associations, mainly the protective role of the HLA-DRB1\*13 haplotype (five amino acid residues of the "wildtype" DERAA at positions 70–74), are shown in European populations. Individuals of such a haplotype have a decreased number of autoreactive T-cells, presumably due to their negative thymus selection [6]. Associations with HLA-DRB1 alleles at position 57 rather than 71–74 are shown in African populations [12]. Thus, the main critical site related to RA development in different populations of both Caucasians and Asians is the 13th amino acid residue. This position substitution increases its pathogenic effect as follows: Ser < Gly ≤ Tyr < Arg < Phe < His [8]

The observed data explain the hypervariable epitope and its polymorphism roles in the development of RA with ACPA formation. However, HLA-RA associations are shown in the absence of ACPAs as well. The DRB1\*03:01 allele associated with ACRA-negative RA was found in Europeans, but not Japanese, in whom HLA-DR8 and DR14 homozygotes are associated with the disease. In addition, DRB1\*03 (serine at position 11) and HLA-B\*08 (aspartic acid at position 9) alleles are associated with ACPA-negative RA regardless of population. Hence, RA is not only clinically but also genetically a heterogeneous disease [13].

In general, RA-associated amino acid positions are located in the middle of the HLA molecules epitope-binding pockets; thus, the substitutions can directly affect antigenic specificity and result in protein binding to the citrulline residues. In discrepancy with the Hardy–Weinberg ratio, heterozygotes are more common in patients with RA (they have a greater predisposition to ACPA-positive RA). This could be due to a wider range of exposed autoantigens compared to homozygotes [14]. Citrullinated proteins are seen as foreign antigens. This complies with the confirmed hypotheses of neoantigens (i.e., peptides with citrulline residues), as well as with the assumption that the calreticulin signalling pathway and the low-affinity HLA-DR molecules carrying SE epitopes for class II-associated invariant chain peptides (CLIPs) are involved in RA pathogenesis [10,15].

In addition to the HLA-DRB1 alleles, other loci have also been associated with RA development, particularly the \*04 allele of the DQB1 locus, as revealed by meta-analysis [16]. It is not only the polymorphisms predisposed to RA that are of interest to professionals in rheumatology, but also those associated with disease prognosis and the severity of the disease. Thus, the HLA-DRB1\*08, HLA-DRB1\*11, and HLA-DPB1\*02:01 alleles are associated with oligoarticular RA—no more than 4 points, according to the International League of Associations for Rheumatology (ILAR) classification. Polyarticular RA (5 points or more) is associated with the HLA-DRB1\*11:01 and HLA-DRB1\*11:04 alleles [10]. At the same time, a meta-analysis of 2775 RA cases showed the association between HLA-DRB1\*04:01 polymorphism and the degree of joint damage [17]. The combination of HLA gene alleles predisposed to RA predetermines the genetic component for the development of this disease. At the same time, many other genes encoding enzymes, receptors, and auxiliary proteins may affect the severity of the clinical manifestations and the development of RA [18].
