Association of rheumatoid arthritis with HLA-DR1 and HLA-DR4 in Hungary.

Susceptibility to and outcome for rheumatoid arthritis (RA) have been associated with particular HLA-DR alleles, but these alleles vary among ethnic groups and geographic areas. The frequency of HLA-DR1 (HLA-DRB1*0101, DRB1*0102) and HLA-DR4 (DRB1*0401, DRB1*0404) alleles is elevated among Caucasian patients with RA. We studied a northeastern Hungarian population of RA patients to determine the frequency of HLA-DR1 and HLA-DR4 phenotypes in this population and to compare it with healthy control subjects, as well as to investigate whether the presence of these alleles could be a marker for RA. We performed HLA-DRB1 genotyping (DRB1*01-DRB1*16) in 83 RA patients and 55 healthy controls using polymerase chain reaction with sequence-specific primers (PCR-SSP). In the case of HLA-DR1- or HLA-DR4-positive patients, the DR1 and DR4 subtypes were also determined. The frequency of HLA-DR4 alleles was significantly higher in RA patients than in controls (31.3 vs. 10.9%; P <.05). HLA-DR1, in particular, tended to be more frequent in patients than in controls (32.5 vs. 18.1%). Among the HLA-DR4 subtypes, DRB1*0401 and DRB1*0404 were the most common alleles found in both groups. However, no significant differences were seen in the frequency of HLA-DRB1*0401 and HLA-DRB1*0404 between RA patients and controls. In contrast, HLA-DRB1*0405 and HLA-DRB1*0408 were significantly more common in RA patients than in control subjects. Among HLA-DR1 subtypes, the DRB1*0101 allele was most commonly detected, but HLA-DRB1*0101 as well as DRB1*0102 and DRB1*0105 were similarly frequent in RA patients and controls. HLA-DR12 was more common among controls than in RA patients (18.1 vs. 0%; P <.05). Our results generally agree with the findings in other Caucasian populations. Nonetheless, we found differences in the frequency of HLA-DR1 and HLA-DR4 subtypes among Hungarian patients compared with reports from other geographic regions (e.g., Finland and Asia). Our data suggest that in northeastern Hungary, HLA-DR4 as well as its subtypes DRB1*0405 and DRB1*0408 may be involved in susceptibility to RA, but HLA-DR1 may not. In addition, the presence of HLA-DR12, at least in Hungary, may protect from this disease.

[Proliferation of type II collagen specific T cell response and antibody formation in rheumatoid arthritis and their relations to HLA-DR4].

OBJECTIVE: To investigate the proliferation of type II collagen specific T cell response and antibody formation in rheumatoid arthritis (RA) and their relations to HLA-DR4 subtype. METHODS: Peripheral blood mononuclear cells and serum were obtained from 62 RA patients, 14 males and 48 females, aged 43 +/- 29. CII263-272 decapeptide and the peripheral blood mononuclear cells (PBMCs) from 62 RA patients were co-incubated for 5 approximately 7 days. MTT method was used to examine the T cell proliferation. The serum antibodies specific to CII 263-272 were examined by ELISA. HLA-DR typing was detected in 40 patients by SSP-PCR. RESULTS: Positive T cell response challenged by CII 263-272 decapeptide was observed in 38 (61.3%) RA patients. The positive rate of CII 263-272 antibody was 66.7% in the T cell response positive group, significantly higher than that of the T cell response negative group (34.6%, P < 0.05). The T cell response rate of the CII 263-272 antibody positive group was 69.7%, significant higher than that of the negative group (42.1%, P < 0.05). In all patients, the stimulation index (SI) of T cell proliferation to CII263-272 peptide was positively correlated with the level of antibody (r = 0.68, P < 0.01). HLA-DR4 allele was detected in 16 out of the 40 patients. Positive T cell response and presence of antibody to CII 263-272 were not associated with HLA-DR4 phenotype. CONCLUSION: The formation of CII antibody may be related to B cell activation mediated by CII specific T cell.

HLA, salivary IgA and mutans streptococci--is there a relation?

The aim of the present studies was to investigate a possible relationship between the human leukocyte antigen (HLA) complex, colonization of mutans streptococci and salivary immunoglobulin A (IgA) antibodies against mutans streptococcal antigens. In the first study a strong inverse relationship between HLA-DR4 and levels of mutans streptococci was observed for a group of renal transplant patients (I). In a group with healthy blood-donors a similar trend was observed (I). This tendency was also seen for a selected population investigated in the second study (II). Since the HLA molecules regulate the production of antibodies in saliva, the salivary IgA activity to three oral streptococci in a population of HLA-DR4-positive and DR4-negative subjects was investigated in the following study (III). It was found that the HLA-DR4-positive subjects, especially the DRB1*0401 and DRB1*0404 subgroups, showed a weaker IgA activity, in particular to Streptococcus mutans, as compared to the HLA-DR4-negative. However, immune response patterns revealed by Western blotting are often complex and for further studies with larger study populations it was crucial to unravel the nature of the detected antigens. In the fourth study (IV), untreated saliva, as well as saliva, in which cell-surface reactive IgA had been absorbed with whole bacteria cells, were analysed in Western blot against different oral streptococci. The high molecular bands, that were absent after absorption, likely represented cell-surface antigens and were thus of interest as they might be involved in adhesion mechanisms and available for blocking in vivo. In the next study (V), the salivary IgA activity to cell-surface antigens of three oral streptococci in relation to different HLA-DRB1*4 alleles was studied in a larger population. The immunoblots were analysed in a computer program and intensity graphs revealed that the DRB1*0401 and *0404 subgroups, compared to other DRB1*04 types, showed fewer as well as less intense immunoblot bands to antigens from S. mutans, S. sobrinus and streptococcal antigen (SA) I/II, but not S. parasanguis. The main conclusion from this thesis is that the HLA profile of the individual seems to influence the salivary IgA response to mutans streptococcal antigens and might thus also affect the conditions for the bacteria in the oral cavity.

[Relation between the clinical course of Vogt-Koyanagi-Harada's disease and human leukocyte antigen (HLA)-DR4 subtypes].

Vogt-Koyanagi-Harada's disease (VKH) is an autoimmune disorder affecting melanocyte-containing tissues. VKH is strongly related to human leukocyte antigen (HLA)-DR4, a heterogeneous HLA specificity consisting of at least 12 different genotypes. The disease has two types: prolonged and non-prolonged, depending upon the clinical course. Fifty-four Japanese patients comprising 27 with the prolonged type, 15 with the non-prolonged type, and 12 new cases of VKH were included in this study. Ninety-three percent of the patients with all types had HLA-DR4. All 27 patients with the prolonged type had either the DRB1*0405 or *0410 genotype, and the non-prolonged type included two DR4-negative and four DRB1*0405 and *0410 negative patients. Our results indicate that the clinical course of VKH is determined partly by the patient's HLA genotype.

Multiplex ARMS-RFLP: a simple and rapid method for HLA-DR4 subtyping.

We have developed a simple and rapid non-radioactive technique for HLA-DR4 subtyping. A multiplex ARMS-RFLP (Amplification Refractory Mutation System--Restriction Fragment Length Polymorphism) system allows HLA-DR4 subtyping by analysis of the products of two multiplex ARMS reaction mixtures. For some cases, restriction enzyme digests (Hae II and/or SacII) of the products are analysed. The technique relies on the fact that an ARMS primer with a mismatch at its 3'-end with respect to the template will not be elongated under PCR conditions. Hence, by designing ARMS primers such that different HLA-DR4 alleles yield PCR products of different lengths, only two reactions, each using a mixture of different ARMS primers, are sufficient to type all of the known HLA-DR4 alleles. This system can distinguish between HLA-DR4 'homozygotes' and 'heterozygotes' since every HLA-DR4 allele can be detected. The ARMS conditions were optimized using DNA from cell lines. This technique has now been used to type a panel of rheumatoid arthritis patients and controls.

PCR-SSO typing for DR4-Dw subtypes: application to unrelated bone marrow transplant donor selection.

Thirty-seven DR4-positive patient-unrelated bone marrow donor pairs previously DR/DQ restriction fragment length polymorphism (RFLP) typed and tested in mixed lymphocyte culture (MLC), have been DR4-Dw subtyped retrospectively using sequence specific oligonucleotide probes. We found that DR4-Dw subtyping substantially increased the accuracy of pre-MLC matching and could potentially accelerate donor searches by avoiding unnecessary MLC tests on Dw-mismatched donors.

Sequence analysis of HLA-DR4B1 subtypes: additional first domain variability is detected by oligonucleotide hybridization and nucleotide sequencing.

The stimulating capacity of HLA-DR4 variants in mixed leukocyte culture correlates with variation in the polymorphic regions of the first domains of their DR beta 1 chains. Variability between amino acids 67 and 86 appears largely to determine HLA-DR4,Dw type. We have used a combination of a DR4B1-specific primer set in the polymerase chain reaction and specific oligonucleotide probes to examine DR4,Dw-associated nucleotide polymorphisms. Phenotype and gene frequencies are reported among 44 normal DR4 Caucasoids. Oligonucleotide probes were selected which enabled definition of Dw4-, w14-, w10-, w13-, and w15-associated sequences. Unexpectedly, several subjects were positive for Dw15 sequences which are usually characteristic of Oriental populations. Dw15 assignment was confirmed by nucleotide sequencing of DR4B1 polymerase chain reaction products. A pair of oligonucleotides informative for the glycine or valine dimorphism at position 86 was used to identify two novel DR4B1 alleles, designated 13.2 and 14.2. Nucleotide sequencing shows that these represent recombinants between third hypervariable regions associated with Dw13 and Dw14 and a codon for glycine at position 86 which is usually found associated with Dw4 and Dw15 sequences.

Shared molecular markers of genetic predisposition to seropositive rheumatoid arthritis.

Rheumatoid arthritis is associated with the human class II major histocompatibility complex antigens known as HLA-DR4. HLA-DR4 can be subdivided by cellular typing into five subtypes: Dw4, Dw10, Dw13, Dw14, and Dw15. By traditional serologic methods, 60-80% of rheumatoid arthritis patients type HLA-DR4 compared to approximately 20% of the general population. It has been demonstrated, using a panel of four alloreactive T-cell clones, each of which recognized HLA-DR4, Dw14 homozygous typing cells, that cells from all of a group of 23 rheumatoid arthritis patients could be recognized by one or more of these clones regardless of the patients' serologic typing. As the expressed polymorphism of the DR molecule is accounted for by the beta 1 gene, this gene was amplified, using the polymerase chain reaction, and sequenced. Seven patients whose cells were recognized by one of the DR4, DW14-specific T-cell clones, T431, were analyzed. All of these patients shared a common sequence in the third hypervariable region of the DR beta 1 chain gene. The sequence identified is the one normally associated with DR4, Dw14 and DR1. Patients and DR4-positive controls whose cells did not stimulate this clone did not share this sequence. These results suggest that this hypervariable region might be an important contribution to a restriction site for the putative causative agent(s) in rheumatoid arthritis.

DNA typing for class II HLA antigens with allele-specific or group-specific amplification. I. Typing for subsets of HLA-DR4.

DNA sequences that distinguish the subsets of HLA-DR4 are also found on several other alleles. This makes typing of heterozygotes with oligonucleotide probes quite impractical. We have therefore developed a procedure in which, in a first step, DNA of the genes to be analyzed is amplified selectively, using group-specific primers. In the case of DR4-DRB1, a primer matching codons 5 to 13 when used in the polymerase chain reaction resulted in products that were entirely suitable for typing for the DR4 subsets. Using appropriate probes, eight distinct subsets were identified. However, only six of them were represented in a normal North American Caucasian panel. In conjunction with a method for rapid DNA extraction, the procedure offers a simple, highly specific and reproducible method for determining subtypes of HLA-DR4 that at present cannot be recognized by serologic methods.