Comparison of HLA class II genes in Caucasoid, Chinese, and Japanese patients with primary Sjögren's syndrome.

To better define the genetic factors that predispose to primary Sjögren's syndrome (SS), we have used polymerase chain reaction in combination with oligonucleotide probe hybridization and DNA sequencing to analyze HLA-DRB1, -DQA1, -DQB1, and -DPB1 alleles in Caucasoid (California), Japanese (Tokyo), and Chinese (Shanghai and Beijing) SS patients. In comparison to local controls in each region, we found: 1) increased frequency of the predicted haplotype HLA-DRB1*0301-DRB3*0101-DQA1*0501-DQB1*0201 in Caucasoid patients (p < 0.001); 2) increased frequency of the predicted haplotype HLA-DRB1*0405-DRB4*0101-DQA1*0301-DQB1*0401 in Japanese patients (p < 0.05); 3) increased frequency of the predicted haplotype DRB1*0803-DQA1*0103-DQB1*0601 in Chinese patients (p < 0.05); and 4) no statistically significant association with DPB1 alleles in any group, although an increased number of Caucasoid and Japanese SS patients possessed DPB1*0301. Comparison of DNA sequences for the three disease-associated haplotypes in these ethnic groups revealed a shared region of predicted amino acids from positions 58 to 69 in the first domain of HLA-DQB1. These results extend previous studies by demonstrating that no single class II allele was associated with 1 degree SS in the different ethnic groups. However, a shared amino acid motif in the DQB1 first domain was present in each disease-associated haplotype.

Antiphospholipid antibodies and HLA associations in primary Sjögren's syndrome.

Blood samples from 65 patients with primary Sjögren's syndrome were evaluated for the presence of antiphospholipid antibodies. Increased levels of antiphospholipid antibodies were found in 13 of 65 (20%) of patients. These antiphospholipid antibodies were predominantly of the IgA isotype, in contrast with the IgG isotype antiphospholipid antibodies found in patients with systemic lupus erythematosus (SLE). The presence of IgA antiphospholipid antibodies in the patients with primary Sjögren's syndrome was not significantly associated with arterial or vascular thrombosis, nor peripheral or central nervous system vasculitis. There was no association with laboratory determined features such as lupus anticoagulant or false positive results of the Venereal Disease Research Laboratory (VDRL) test. Oligonucleotide specific DNA amplification and hybridisation with allele specific probes was used to examine the HLA-D antigens occurring in this group of patients with primary Sjögren's syndrome. Of 13 patients with antiphospholipid antibodies, seven had the genotype HLA-DR2/DR3. However, compared with the whole group of 65 patients with Sjögren's syndrome, no increased occurrence of haplotype DR2 or DR3 was noted. These results suggest that gene interaction between DR2 and DR3 may play a part in the production of antiphospholipid antibodies in patients with Sjögren's syndrome. In contrast with patients with SLE, the IgA antiphospholipid antibodies in patients with Sjögren's syndrome are not risk factors for thrombosis or vasculitis. The presence of IgA antiphospholipid antibodies in patients with Sjögren's syndrome probably reflects its production at mucosal sites of inflammation and the absence of vasculopathy may be due to the inability of IgA antibodies to activate complement.

Specific HLA-DQA and HLA-DRB1 alleles confer susceptibility to Sjögren's syndrome and autoantibody production.

Primary Sjögren's syndrome (1. SS) is a systemic autoimmune disease characterized by lymphocytic infiltration of the salivary glands and autoantibody production. In order to identify genetic factors that play a role in pathogenesis and predict extent of disease, we used Southern blot and polymerase chain reaction (PCR) methods to detect polymorphisms of the HLA-DRB1 (DR), HLA-DRB3 (DRw52), and HLA-DQA1 genes among 75 Caucasoid 1. SS patients and 150 Caucasoid controls living in the same geographic region of Southern California. We found significantly increased frequency of HLA-DR3 (P less than .001), HLA-DW52a (P less than .001), and HLA-DQA4 (P less than .05), in comparison to normal controls. Also, an increased frequency of heterozygosity for HLA-DQA1/DQA4 (P less than .05) was present among 1. SS patients. Autoantibodies to SS-A and to SS-B were significantly associated with DR3 (P less than .001), HLA-DQA4, (P less than .05), and DQA4/DQA1 heterozygotes (P less than .01). Among the 1. SS patients, clinical and laboratory features such as hypergammaglobulinemia, symmetric peripheral neuropathy, and hypothyroidism were significantly associated with HLA-DR3 (P less than .01) but not with HLA-DR2 (P greater than .10). In comparison, 1. SS patients with leukocytoclastic vasculitis were more frequently HLA-DR2 (P less than .05). These results using PCR methods confirm and extend prior studies that have used serologic methods.

Use of DNA amplification methods for clinical diagnosis in autoimmune diseases.

Autoimmune disease is generally felt to result from the interaction of genetic and environmental factors. In recent years, significant advances have been made in using recombinant DNA methods to analyze specific genetic factors and infectious agents. However, new techniques are needed that are more rapid, inexpensive, and suitable for small tissue biopsies obtained early in the course of disease. New methods of DNA amplification based on polymerase chain reaction (PCR) and Q beta-replicase (Q beta R) have recently been reported. These methods are briefly reviewed, and their potential applications to patients with autoimmune disease are presented. Several types of applications can be considered, including detection of: a) specific HLA-D alleles in order to predict prognosis and better utilize existing medications; b) bacterial, fungal, and spirochete infections in joint aspirates or synovial biopsies; c) human immunodeficiency virus (HIV) and other viruses (e.g., EBV, CMV) that may be associated with immune dysregulation in certain patients; and d) neoplastic transformation in blood or tissues by determining monoclonal gene rearrangements, karyotypic alterations or oncogene activation. It is likely that routine clinical laboratories will soon begin implementing DNA amplification methods in order to screen blood products for infectious agents (especially HIV and hepatitis B virus). Because these techniques will be readily available, rheumatologists/clinical immunologists should begin developing strategies that will allow them to use these methods in a cost-effective manner for diagnosis and monitoring treatment.

A family with an apparent HLA-DR triplet: evidence for exchange of functional HLA-DR beta-genes between different haplotypes.

Serological and protein analysis showed that HLA-DR1 and DR2 short (s) segregated together as one haplotype, resulting in a HLA-DR triplet as found in a family study. Both DR1 and DR2 molecules were coordinately expressed and were shown to function as restriction elements in antigen presentation assays. This unique HLA-DR1, 2s haplotype was further studied by Southern blot analysis. Based upon well-known restriction fragment length polymorphisms for the involved gene sets, i.e. DR1 and DR2 along with the DR type from the other haplotype, the genes as identified by restriction fragment length polymorphism of the triplet could be established. Pseudogenes, which are included in the previously described gene sets of HLA-DR1 and DR2 are apparently lacking in the triplet. We therefore postulate that during an unequal crossing-over event the DR beta-pseudogene of DR2 could be exchanged by a functional DR1 beta-gene.

HLA-linked control of predisposition to lepromatous leprosy.

In order to investigate if genes conferring susceptibility to leprosy and/or predisposition to a particular leprosy type are linked to HLA, we performed HLA-A and HLA-B typing on members of 29 families containing at least two siblings affected with leprosy from a leprosy endemic area in Jiangsu Province, People's Republic of China. Moreover, we performed a lepromin test in nearly all of the members of eight families containing at least two siblings affected with lepromatous leprosy. For 26 families the data permitted analyses of the segregation of parental HLA-haplotypes observed among children in relation to leprosy status. Siblings affected with lepromatous (LL or BL) leprosy shared parental HLA-haplotypes significantly more often than expected (p less than 0.05), and a highly significant deficit (p less than 0.0005) of shared HLA-haplotypes was observed among affected siblings discordant for leprosy type. These data confirm the HLA-linked control of leprosy type and, in particular, recently obtained evidence for linkage of predisposition to lepromatous leprosy with HLA. Healthy siblings did not share a haplotype more often than expected, and those haplotypes which were shared among all leprosy patients of a sibship did not occur less frequently than expected among the healthy siblings of the same sibship. The latter data confirm the absence of linkage of genes conferring susceptibility or resistance to leprosy with HLA. An analysis of the lepromin test results observed among healthy siblings showed no evidence for cosegregation of the results with HLA.