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An obvious way to unravel the apparently complex association between human leukocyte antigen (HLA) and rheumatoid arthritis (RA) is to reduce the heterogeneity of this multifactorial disease. Recently we have discovered that shared epitope (SE)-positive HLA-DRB1 alleles are exclusively associated with a subgroup of RA patients that test positive for auto-antibodies against cyclic citrullinated peptides. Further studies suggested that SE-positive alleles are classical immune response genes for the development of these antibodies. On the basis of these and other data we formulated a two-hit model for the pathogenesis of RA which incorporates a novel "citrullinated" SE hypothesis. About 5 years ago Zanelli et al. reported that HLA-DR6 (*1301 and *1302) and some other DR alleles that share the DERAA-sequence on amino acids 70-74 of their third hypervariable region are associated with protection from (severe) RA. Recently we corroborated these data in a large prospective cohort study and demonstrated that protection was observed both in the presence and in the absence of a SE susceptibility allele on the other haplotype. Finally we review the state of the art of the association of noninherited maternal HLA antigens with both susceptibility to and protection from RA.

Sequencing of alleles of the highly polymorphic, multiple loci HLA-DRB gene family was performed by pyrosequencing using purified DNA from the 11(th) International Histocompatibility Workshop human lymphoblastiod cell lines as well as genomic DNA isolated from blood samples obtained from healthy adult volunteers. Genomic DNA was prepared from donors whose blood had been stored either frozen or as dried blood spots. Pyrosequence-based typing was optimized for identifying alleles of the HLA-DRB1, -3, -4, and -5 genes. The procedure should be applicable to other HLA loci including the class I genes HLA-A and -B that, along with HLA-DRB, are crucial for histocompatibility matching of tissue antigens during transplantation. Computer simulation of pyrosequencing data suggest that alleles of HLA-DRB1, -3, -4, and -5 were readily identifiable by pyrosequencing as were their heterozygous allelic combinations. Pyrosequencing primers were designed to specifically sequence HLA loci of interest even in a background of other amplified, closely related sequences such as alleles of the pseudogene HLA-DRB6, -7, -8, and -9. Polymorphic residues of HLA-DRB genes were identified within each pyrosequencing reaction, obtained by 50 to 70 nucleotide read lengths. Heterozygous allelic combinations of HLA genes were analyzed and compared successfully to genotyping of alleles by sequence-specific oligonucleotide probe hybridization as well as allele specific polymerase chain reaction protocols. Pyrosequence-based typing is compatible with genotyping of allelic combinations expected from heterozygous individuals, resulting in nucleotide resolution of the highly polymorphic HLA system. Using a single pyrosequence instrument, complete typing of HLA-DRB genes can be performed daily on hundreds of individuals for high resolution histocompatibility genotyping studies.

The HLA-DR4 haplotype consists of four DRB genes: DRB1*04, DRBVII, DRBVIII, and DRB4*01, arranged in this order on the chromosome. The DRB1 and DRB4 genes code for beta chains of the alpha beta heterodimers expressed on the cell surface and bearing the HLA-DR4 and HLA-DRw53 determinants, respectively; the DRBVII and DRBVIII are pseudogenes. We found and sequenced a gene closely related to HLA-DRBVIII in the genome of the lowland gorilla "Sylvia." We designate this gene Gogo-DRB8. The close relationship between the two genes is indicated by the overall sequence similarity, the absence of recognizable exons 1 and 2 in both genes, the presence of two Alu repeats at corresponding positions, and high sequence similarity between corresponding repeats. The comparison with an outgroup (tamarin) gene and the functional counterparts of the DRB8 gene indicate that DRB8 emerged between 18 and 26 million years ago and became inactivated at the same time as or shortly after its creation. Hence DRB8 has probably existed as a pseudogene since the divergence of apes from Old World monkeys more than 20 million years ago.