The HLA system and its contribution to the genetics of rheumatic diseases

The results of the study of histocompatibility antigens at loci A, B and Dr in patients with RA and SLE, and their first degree relatives are presented. HLA antigens B12. B18, B27, Dr2 and Dr4 were associated with RA. The antigens HLA A11, B7, B35, Dr2 and Dr3 were associated with SLE. The influence of HLA antigens on formation of clinical picture of RA and SLE was determined. Evaluation of interallelic and interloci antigens interaction in a relative risk of disease suggests that, in some cases, there is a "superdominance" effect. Some combinations of HLA antigens at loci B and Dr increase the disease risk for RA and SLE. Analysis of test-marker linkage to genes predisposed to RA and SLE provides no direct confirmation of the hypothesis of their location on the short arm of the sixth chromosome between loci B and Dr, though this possibility cannot be completely excluded.     

Analysis of families in the multiple autoimmune disease genetics consortium (MADGC) collection: the PTPN22 620W allele associates with multiple autoimmune phenotypes

Autoimmune disorders constitute a diverse group of phenotypes with overlapping features and a tendency toward familial aggregation. It is likely that common underlying genes are involved in these disorders. Until very recently, no specific alleles--aside from a few common human leukocyte antigen class II genes--had been identified that clearly associate with multiple different autoimmune diseases. In this study, we describe a unique collection of 265 multiplex families assembled by the Multiple Autoimmune Disease Genetics Consortium (MADGC). At least two of nine "core" autoimmune diseases are present in each of these families. These core diseases include rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), type 1 diabetes (T1D), multiple sclerosis (MS), autoimmune thyroid disease (Hashimoto thyroiditis or Graves disease), juvenile RA, inflammatory bowel disease (Crohn disease or ulcerative colitis), psoriasis, and primary Sjogren syndrome. We report that a recently described functional single-nucleotide polymorphism (rs2476601, encoding R620W) in the intracellular tyrosine phosphatase (PTPN22) confers risk of four separate autoimmune phenotypes in these families: T1D, RA, SLE, and Hashimoto thyroiditis. MS did not show association with the PTPN22 risk allele. These findings suggest a common underlying etiologic pathway for some, but not all, autoimmune disorders, and they suggest that MS may have a pathogenesis that is distinct from RA, SLE, and T1D. DNA and clinical data for the MADGC families are available to the scientific community; these data will provide a valuable resource for the dissection of the complex genetic factors that underlie the various autoimmune phenotypes.     

Sex influences on the penetrance of HLA shared-epitope genotypes for rheumatoid arthritis.

The association between rheumatoid arthritis (RA) and HLA DRB1 alleles may arise through linkage disequilibrium with a disease locus or the direct involvement of HLA alleles in RA. In support of the latter possibility, the shared-epitope hypothesis has been postulated, stating that conformationally similar DR beta chains encoded by several DRB1 alleles confer disease susceptibility. To examine these alternative hypotheses of marker-disease association and to investigate gender differences in RA susceptibility, we analyzed the distributions of PCR-based DRB1 genotypes of 309 Caucasian RA patients and 283 Caucasian controls. Initially, the marker-association-segregation chi 2 method was used to evaluate evidence for linkage disequilibrium and the direct involvement of markers DR4 Dw4, DR4 Dw14, and DR1 in RA susceptibility. Additional shared-epitope models that grouped DRB1 alleles into five classes (*0401, *0404/*0102, *0405/*0408/*0101, *1001, and all others) and postulated relationships between genotypes and RA susceptibility were also fitted to observed genotypic distributions by the method of minimal chi 2. For females, a linkage-disequilibrium model provided a good fit to the data, as did a shared-epitope model with RA most penetrant among individuals with the *0401,*0401 genotype. For males, the best model indicated highest RA penetrance among shared-epitope compound heterozygotes. Clinically, male RA patients had more subcutaneous nodules and greater use of slowly acting antirheumatic drugs, while female RA patients had earlier disease onset. This study therefore suggests that sex-related factors influence the RA penetrance associated with DRB1 shared-epitope genotypes and that DRB1 effects on RA prognosis and pathogenesis should be considered separately for men and women.     

Genetic interrelationship between insulin-dependent diabetes mellitus, the autoimmune thyroid diseases, and rheumatoid arthritis.

To investigate the possible coinheritance of autoimmune diseases that are associated with the same HLA antigen, we studied 70 families in which at least two siblings had either type I diabetes mellitus (IDDM), autoimmune thyroid disease (ATD), rheumatoid arthritis (RA), or a combination of these diseases. HLA-A, B, and C typing was performed on all affected sibs in one generation or more. First, we estimated by sib-pair analysis the disease allele frequency (pD) and the mode of inheritance for each disease. According to the method of ascertainment entered into the analysis, the pD for ATD ranged from .120 to .180, for an additive (dominant) mode of inheritance. For RA, the pD ranged from .254 to .341, also for additive inheritance, although recessive inheritance could not be excluded. For IDDM, the pD ranged from .336 to .337 for recessive inheritance; additive inheritance was rejected. Second, we examined the distribution of shared parental haplotypes in pairs of siblings that were discordant for their autoimmune diseases. The results suggested that the same haplotype may predispose to both IDDM and ATD, or IDDM and RA, but not to both RA and ATD. Analysis of pedigrees supported this hypothesis. In 16 families typed for HLA-DR also, the haplotype predisposing to both IDDM and ATD was assigned from pedigree information to DR3 (44%), DR4 (39%), or DR5, DR6, or DR7 (5.5% each). In some families, these haplotypes segregated over several generations with ATD only (either clinical or subclinical), suggesting that in such families, ATD was a marker for a susceptibility to IDDM. In several families, an IDDM haplotype segregated with RA but not with ATD. This suggests that ATD- and RA-associated susceptibilities to IDDM may be biologically different and thus independently increase the risk of IDDM.     

Association of MHC and rheumatoid arthritis. Regulatory role of HLA class II molecules in animal models of RA: studies on transgenic/knockout mice

Human leucocyte antigen (HLA) class II molecules have been shown to be associated with predisposition to rheumatoid arthritis (RA). We generated HLA-DR and DQ transgenic mice that lacked endogenous class II molecules to study the interaction between the DR and DQ molecules and define the immunologic mechanisms in rheumatoid arthritis. Using collagen-induced arthritis (CIA) as an experimental model for inflammatory polyarthritis, we show that both DQ and DR are involved in predisposition or resistance to arthritis. Our studies suggest that polymorphism in DQB1 genes may determine predisposition to RA while the DRB1 polymorphism may dictate severity/protection of the disease. These mice provide powerful tools to develop immunotherapeutic protocols.     

Association of MHC and rheumatoid arthritis: Regulatory role of HLA class II molecules in animal models of RA - studies on transgenic/knockout mice

Human leucocyte antigen (HLA) class II molecules have been shown to be associated with predisposition to rheumatoid arthritis (RA). We generated HLA-DR and DQ transgenic mice that lacked endogenous class II molecules to study the interaction between the DR and DQ molecules and define the immunologic mechanisms in rheumatoid arthritis. Using collagen-induced arthritis (CIA) as an experimental model for inflammatory polyarthritis, we show that both DQ and DR are involved in predisposition or resistance to arthritis. Our studies suggest that polymorphism in DQB1 genes may determine predisposition to RA while the DRB1 polymorphism may dictate severity/protection of the disease. These mice provide powerful tools to develop immunotherapeutic protocols.     

The polymorphisms of HTC-defined HLA specificities in the Shanghai Chinese population

With reference sera and homozygous typing cells (HTCs) of 3rd Asia-Oceania Histocompatibility Workshop Conference, 56 healthy unrelated subjects in Shanghai were typed for HLA-A, B, C, DR, DQ, and Dw. This paper presents the results of HLA-Dw typing, its relationship to serological class II antigens, and the distribution of Dw in the population. The polymorphism patterns of Chinese Dw specificities were quite different from those in Caucasoids and Japanese. The predominant Dw phenotypes detected in Shanghai Chinese were Dw 2, Dw 3, DKT 2, Dw 7 c, (Dw7 + Dw 17) and Dw 23 (DB 5). And significant correlations were observed between Dw 1 and DR 1, Dw 2 and DR 2, Dw 3 and DR 3, Dw 7 c and DR 7, DB 7 and DRw 8, as well as Dw 23 and DR 9. SMY 129, a novel Dw specificity defined by local HTCs and co-studied by the laboratories joined for Dw typing in 3rd AOHWC showed its correlation with DR 5. Nevertheless, more than fifty percent of Dw specificities could not be assigned in the four correspondent designated serological antigens, DR 2, DR 5, DRw 8 and DR 9, respectively, which, together with other blank Dw specificities, gave a total blank Dw gene frequency as high as 43.2% in the population. It was suggested by further analysis that novel Dw specificities might be identified more effectively if efforts would be concentrated on DR 5 and DR 9, two antigen families which, in some way, might represent the characteristics of HLA system in Chinese. Besides, certain HTC-defined antigens, e.g. Dw 3 and the DR 4-related Dw specificities, have been revealed to be in linkage disequilibrium with other DR antigens in addition with the correspondent designated ones, resulting in some unique haplotype combinations in Shanghai Chinese. It seems to us that the particular patterns of polymorphisms of serum- and cell-defined HLA class II antigens would be helpful to elucidate the mechanisms by which certain diseases are in association with HLA in Chinese in a different manner as compared with that in Caucasoids.     

HLA antigens and acetylcholine receptor antibodies in penicillamine induced myasthenia gravis.

Antibodies to the acetylcholine receptor and HLA antigens have been studied in patients with myasthenia gravis occurring in association with penicillamine treatment. The properties of the antiacetylcholine receptor in these patients differed from those in patients with idiopathic myasthenia gravis in terms of specificity and affinity. These patients had an increased prevalence of HLA Bw35 and DR1 compared to controls and a decreased frequency of B8 and DR3 compared to patients with idiopathic myasthenia gravis. Likewise, they had a decreased frequency of DR4 compared to patients with rheumatoid arthritis. These data provide supportive evidence for a role for penicillamine in the induction of myasthenia gravis in genetically predisposed individuals.     

Increased incidence of faecal Klebsiella pneumoniae in patients with HLA B7 CREG antigen and men with rheumatoid arthritis.

In a study of the carriage of faecal Klebsiella pneumoniae in 106 patients with rheumatoid arthritis the incidence of carriage was higher in men (28%) than women (14%) (p < 0.001) and klebsiellae were isolated on two or more occasions from a higher proportion of men than women (p < 0.002). The incidence of carriage was increased among patients with rheumatoid arthritis who had B7 cross-reacting (B7 CREG) antigens (32% v 13%--p < 0.001). Carriage of klebsiellae was not associated with clinical disease activity, raised erythrocyte sedimentation rate, drug treatment, or the presence of HLA Dw4 or DRw4 or both. Thus the carriage and prevalence of faecal Klebsiella pneumoniae appear to be partly determined by the sex and HLA state of the host.     

HLA-DR genotype risks in seropositive rheumatoid arthritis.

We studied the distribution of HLA-A, B, C, and -DR antigens in 77 Caucasian patients with sero-positive rheumatoid arthritis. Forty-four patients were genotyped and compared with the control panel of 110 unrelated Caucasian genotyped donors. The data obtained confirm the association of DR4 with RA, and reveal an increased risk of disease for patients carrying DR1, DR2, and DR3, compared to the risk for those carrying other antigens, such as DR5, DRw6, and DR7. There is a higher risk for DR4/4 homozygotes than for DR4/1, DR4/2, or DR4/3 heterozygotes. DR4/5, DR4/6, and DR4/7 have a lower risk than the previously mentioned genotypes. The genotype risks are compatible with the inheritance of a single, linked genetic determinant of disease susceptibility, but we are unable to distinguish between recessive and dominant inheritance of susceptibility using the "antigen-frequencies-amongst-diseases" method. DR4 seems to be more frequent in patients in whom onset occurs before the age of 35 (79% vs. 54% DR4 positive). A significant excess of DR3 + is observed in patients with toxic complications following treatment with gold salts (X2(1) = 8.96).     

Frequency of HLA antigens in patients with psoriasis or psoriatic arthritis.

A study of 138 patients with psoriasis--74 with psoriasis alone and 64 with psoriatic arthritis--revealed a significantly increased frequency of the HLA antigens A1, A28, B13, DR7 and MT3 in those with psoriasis alone and of Bw39 in those with psoriatic arthritis. The frequency of B17 was higher in both patient groups than in a control group of healthy individuals. The frequency of DRw6 was slightly higher in the patients with psoriasis alone (17.8%) than in the controls (4.7%), and that of DR7 was higher in the patients with psoriatic arthritis (52.9%) than in the controls (32.6%). Elevated levels of serum IgG and IgA along with positive results of tests for antinuclear antibody or rheumatoid factor or both were present in less than a tenth of the patients with psoriatic rash alone and in up to a third of those with psoriatic arthritis. Psoriatic arthritis was found to be less likely to develop in patients with purely guttate psoriasis than in those with other types of psoriasis. Clinical subtypes of psoriatic rash or psoriatic arthritis were not associated with the presence of particular HLA antigens.     

Investigation of the HLA component involved in rheumatoid arthritis (RA) by using the marker association-segregation chi-square (MASC) method: rejection of the unifying-shared-epitope hypothesis.

In order to investigate the HLA component involved in rheumatoid arthritis (RA), we tested genetic models by the marker association-segregation chi 2 (MASC) method, using the HLA genotypic distribution observed in a sample of 97 RA patients. First we tested models assuming the involvement of a susceptibility gene linked to the DR locus. We showed that the present data are compatible with a simple model assuming the effect of a recessive allele of a biallelic locus linked to the DR locus and without any assumption of synergistic effect. Then we considered models assuming the direct involvement of the DR allele products, and we tested the unifying-shared-epitope hypothesis, which has been proposed. Under this hypothesis the DR alleles are assumed to be directly involved in the susceptibility to the disease because of the presence of similar or identical amino acid sequences in position 70-74 of the third hypervariable region of the DRBI molecules, shared by the RA-associated DR alleles DR4Dw4, DR4Dw14, and DR1. This hypothesis was strongly rejected with the present data. In the case of the direct involvement of the DR alleles, hypotheses more complex than the unifying-shared-epitope hypothesis would have to be considered.     

Dissecting the genetic complexity of the association between human leukocyte antigens and rheumatoid arthritis

Rheumatoid arthritis (RA) is an inflammatory disease with a complex genetic component. An association between RA and the human leukocyte antigen (HLA) complex has long been observed in many different populations, and most studies have focused on a direct role for the HLA-DRB1 "shared epitope" in disease susceptibility. We have performed an extensive haplotype analysis, using 54 markers distributed across the entire HLA complex, in a set of 469 multicase families with RA. The results show that, in addition to associations with the DRB1 alleles, at least two additional genetic effects are present within the major histocompatibility complex. One of these lies within a 497-kb region in the central portion of the HLA complex, an interval that excludes DRB1. This genetic risk factor is present on a segment of a highly conserved ancestral A1-B8-DRB1*03 (8.1) haplotype. Additional risk genes may also be present in the HLA class I region in a subset of DRB1*0404 haplotypes. These data emphasize the importance of defining haplotypes when trying to understand the HLA associations with disease, and they clearly demonstrate that such associations with RA are complex and cannot be completely explained by the DRB1 locus.     

The clinical immunogenetics of viral hepatitis C in a Caucasoid population of western Siberia

The analysis of the immunogenetic studies on hepatitis C patients among the Caucasoid population of western Siberia has revealed a significant increase in the detection rate of antigens HLA-A10 and HLA-DR5, the combinations of DR2-DR5, DR5-DR7, DR1-B27 and the complete absence of antigen HLA-DR4, which is indicative of the fact that susceptibility and resistance to the development of the disease is associated with the genes of the main histocompatibility complex. In hepatitis of mixed etiology, B and C, a significant increase in the occurrence of HLA antigens: -A1, -B8, -DR1 and -DR3, as well as the combinations of A1-DR1, A1-DR3, A3-DR3, A9-A10, DR1-DR3, B8-DR3 is noted; at the same time a decrease in the occurrence of antigen DR4 and its combination with antigen HLA-A2 is observed.     

Production of autoantibodies against citrullinated antigens/peptides by human B cells

Autoantibodies against citrullinated protein Ags (ACPA) are associated with the development of rheumatoid arthritis (RA). This immune response against citrullinated protein Ags, which is thought to be facilitated by certain MHC HLA-DR alleles, is highly specific for this disease and has been speculated to be involved in the pathogenesis. We have previously studied cultures of B cells for the production of Abs against HLA Ags. The aim of the current study was to examine the role of B cells in the production of ACPA in patients with RA. Peripheral blood B cells from RA patients and healthy people were cultured with EL4-B5, a murine cell line expressing human CD40L, and with T cell factors to stimulate the in vitro production of Abs by B cells isolated from peripheral blood. ACPA were produced by cultured B cells from RA patients, as determined by reactivity to cyclic citrullinated peptide (CCP). The results showed that 22% of the healthy persons tested also had B cells that could produce ACPA. Patients with HLA-DR alleles carrying the RA-associated shared epitope appeared to have more B cells with autoimmune potential for CCP than those without such HLA alleles (odds ratio 8.1, p = 0.001). In healthy individuals, anti-CCP-producing B cells were also observed more frequently if the RA-associated MHC genes were present (odds ratio 8.0, p = 0.01). Analysis of B cells in cultures may shed light on the interaction of genetic and environmental factors in the development of RA.     

Nontraditional approaches to biomedical screening: Communication II

On the basis of theoretical studies and literature data, biochemical markers of multifactorial diseases (such as presenile dementia (PD) or Alzheimer’s disease (AD), multiple sclerosis (MS), and rheumatoid arthritis (RA), reflecting the pathogenetic features of these diseases have been systematized. Amyloid β is regarded as a common marker for PD (AD) and RA (along with the BB isozyme of creatine kinase, transaminase, α and -β secretases, acetylcholine, choline acetyltransferase, and acetylcholine esterase); myelin basic protein, tumor necrosis factor α, cephalin, and linoleic acid, as markers of MS; and circulating immune complexes, rheumatoid factor (RF IgG), granzyme A, perforin, acid phosphatase, and cathepsins, as markers of RA. Their contents in biological material obtained from patients were significantly higher or lower than in similar material from healthy subjects. Changes in the levels of some markers in patients were correlated with the severity of the corresponding diseases. Arguments are presented for the possibility of nontraditional biomedical screening of potential means of “molecular biochemical treatment” of these diseases by testing biologically active compounds for these markers.     

Association of HLA DR1 with high D-penicillamine binding to monocytes in females

Binding of D-Penicillamine (D-Pen) to human monocytes was examined by flow cytometry with fluorescent D-Pen conjugate. Cells from HLA DR1-positive healthy females bound significantly more D-Pen than cells from DR1-negative healthy females (P = 0.015), and DR1 was associated with the highest binding among HLA DR antigens. In contrast, DR4 was associated with the lowest binding in healthy females. A difference in D-Pen binding between healthy females who were DR1-positive, DR4-negative and those who were DR1-negative, DR4-positive was statistically significant (P = 0.026). Neither healthy females nor healthy males showed significant associations of D-Pen binding with HLA A, B, or C antigens, nor did healthy males show an association between strength of D-Pen binding and any DR antigens.     

Genetic variability in the tumor necrosis factor-lymphotoxin region influences susceptibility to rheumatoid arthritis.

The major histocompatibility complex class III tumor necrosis factor-lymphotoxin (TNF-LT) region (6p21.3) was investigated as a possible susceptibility locus for rheumatoid arthritis (RA). Inheritance of five TNF microsatellite markers was determined in 50 multiplex families. Overall, 47 different haplotypes were observed. One of these, the TNF a6, b5, c1, d3, e3 (H1) haplotype, was present in 35.3% of affected, but in only 20.5% of unaffected, individuals (P < .005). This haplotype accounted for 21.5% of the parental haplotypes transmitted to affected offspring and only 7.3% not transmitted to affected offspring (P = .0003). The TNF a6 and TNF c1 alleles were individually associated with RA (P = .0005 and .0008, respectively), as were the HLA-DRB1 "shared epitope" (SE) (P = .0001) and HLA-DRB1*0401 (P = .0018). Both univariate and bivariate conditional logistic regression analysis showed significant effects of TNF c1 and SE in increasing risk to RA (P < .001). Stratification by the presence of SE indicated an independent effect of the TNFc1 allele (P = .0003) and the HLA A1, B8, DR3 extended haplotype (always TNFa2, b3, c1, d1, e3) (P = .0027) in SE heterozygotes, while the H1 haplotype was associated with RA in SE homozygotes (P = .0018). The TNF-LT region appears to influence susceptibility to RA, distinct from HLA-DR.     

PHA-T cells in systemic lupus erythematosus and in rheumatoid arthritis: abnormalities in HLA class II antigen induction and in autologous mixed lymphocyte reactions

Analysis of T cells from patients with systemic lupus erythematosus (SLE) and with rheumatoid arthritis (RA) identified a deficit in the induction of HLA Class II antigens by PHA although the proliferative response was normal and in the [3H]thymidine incorporation in autologous mixed lymphocyte reactions (MLR) with PHA-T cells as stimulators. In RA these abnormalities were more marked in patients with active disease than in those in clinical remission. The deficit of autologous MLR with PHA-T cells was more marked than that of autologous MLR with non-T cells and of allogeneic MLR. Serum from patients with SLE and with RA did not display any detectable inhibitory activity on the induction of HLA Class II antigens by PHA, on the proliferative response of lymphocytes to PHA, on autologous MLR with PHA-T cells and with non-T cells as stimulators and on allogeneic MLR. These results suggest that the abnormalities we have identified reflect an intrinsic defect of T cells.