HLA-DR Haplotypes Research Articles

HLA‐D Region genes associated with autoantibody responses to histidyl‐transfer RNA synthetase (Jo‐1) and other translation‐related factors in myositis

Myositis has been associated with HLA-B8 and DR3, especially in white patients with polymyositis and serum anti-Jo-1 antibodies. Twenty-eight patients with myositis and serum translation-related autoantibodies anti-Jo-1, anti-PL-7, anti-PL-12, anti-KJ, and anti-SRP were studied for HLA class II specificities by Southern blotting with HLA-DR beta, DQ beta, and DQ alpha probes. The association of HLA-DR3 (DRw17) with anti-Jo-1 antibodies in white myositis patients was confirmed (P = 0.003, relative risk 8.9). However, HLA-DRw52 haplotypes, regardless of subtype, were present in all of the white and black patients with serum anti-Jo-1 and other translation-related autoantibodies. Moreover, one anti-Jo-1 positive patient had HLA-DRw8, an HLA-DRw52 haplotype on which the DR beta 3 gene has been partially deleted. No HLA-DQ specificity or allele was common to all patients. The HLA-DR3, DR5, DRw6, and DRw8 haplotypes, which bear the HLA-DRw52 specificity, share the most homology in the DR beta 1 first hypervariable region at amino acid positions 9-13. Thus, this DR beta 1 region appears to be the most likely candidate "epitope" for translation-related autoimmune responses in inflammatory myositis.

New HLA‐DR2‐related specificities detected in South African Blacks and individuals of mixed ancestry

This study describes a new HLA-DR2-related specificity DR2LUM (CT) present in South African Blacks and individuals of mixed ancestry (Cape Coloureds). It can be distinguished from the "classic" DR2 specificities. DRw15 and DRw16, using serological and Southern blot techniques. Although no HLA-Dw specificity could be assigned to the DR2LUM(CT) cells, borderline typing reactions with Dw2 HTCs were observed. Southern blot analysis using a DRB probe and the TaqI enzyme has shown that DR2LUM(CT) shared a 1.6 kb fragment with DRw15 and a 4.7 kb fragment with DR1 and DRw10, indicating sequence homology between DR2LUM(CT) and these alleles. In addition, another unusual HLA-DR2 haplotype was found. The DR antigen was typed serologically as DRw16 but showed a combination of restriction fragments which are associated with both the DRw15 and DRw16 specificities. This study demonstrates the value of investigating non-Caucasoid populations in further characterizing the polymorphisms of the HLA class II genes.

DNA restriction fragment length polymorphism of HLA-DR2 haplotypes in normal individuals and in patients with rheumatoid arthritis.

A strong association between HLA-DR4 and rheumatoid arthritis (RA) has been found in a number of populations. In contrast, the incidence of DR2 is decreased in patients with RA, suggesting that this specificity may confer some protection against the disease. A number of subtypes of DR2 have been defined by serology, by responses in mixed lymphocyte culture reaction, and, more recently, by restriction fragment length polymorphism. These subtypes of DR2 are in linkage disequilibrium with different subspecificities of DQw1. It is thus likely that the distribution of these subtypic DR,DQ haplotypes in DR2 positive patients with RA may be important in understanding the genetic basis of susceptibility/resistance to RA. In this paper a study of the subtypes of DR2,DQw1 haplotypes in 18 patients with RA, who required sodium aurothiomalate as a disease remitting drug, and unrelated healthy individuals is reported. Three subtypes of DR2 haplotypes, DRw15 (Dw2),DQw1.2(DQw6), DRw15(Dw12),DQw1.12(DQw6), and DRw16(Dw21),DQw1, AZH (DQw5), were analysed with a cDNA probe for the DQ beta gene. The data show that DR2 positive patients with RA carried either the DRw15(Dw2),DQw6 or DRw15(Dw12),DQw6 haplotype. No patient with RA was positive for the DRw16(Dw21),DQw5 subspecificity. In contrast, six of 29 (21%) normal healthy DR2,DQw1 positive individuals carried the DRw16(Dw21),DQw5 haplotype. These data together with earlier results on the distribution of the DR4,DQw7 haplotype in patients with RA support the hypothesis that DQB1 chain polymorphism may be important in determining susceptibility to severe RA.

Celiac disease is associated with an extended HLA-DR3 haplotype which includes HLA-DPw1

HLA-DP polymorphism was examined among celiac disease patients and controls. Restriction fragment length polymorphism (RFLP) genotyping showed a significant association of DPw1 in celiac disease (32/80) compared with controls (6/53, p = 0.00002). DPw1 typing by RFLP was verified using DPB1-amplified DNA and an oligonucleotide probe specific for the DPw1-associated DPB1 gene. The RFLP-assigned DPw1 genotype corresponded closely to the binding pattern of the sequence-specific oligonucleotide probe, although discrepancies did occur. The association between celiac disease and DPw1, however, remained. Oligonucleotide probe specificity was confirmed by sequencing DPB1-amplitude DNA from four DPw1-genotyped celiacs. DPw1 is only present in celiacs who genotype DR3a-positive. Of DR3a controls 24% are DPw1-positive compared with 5% of non-DR3a controls (p = 0.03), suggesting that an extended DR3a, DPw1 haplotype occurs in the control population. This haplotype forms a large proportion of the DR3a haplotypes predisposing to celiac disease. Alternatively, DPw1 may represent an independent risk factor inherited in linkage with HLA-DR3 and DQw2. Although predisposition to celiac disease is likely to be mediated by a specific DQα/DQβ heterodimer, a direct role for the DPw1 antigen cannot be discounted.

Complotypes in individuals of African origin: frequencies and possible extended MHC haplotypes

We analyzed the frequency distribution of 106 complotypes [four allele sets of the major histocompatibility complex (MHC) genes for the complement proteins factor B, C2, C4A, and C4B] from 32 Black families residing in Boston and Washington, DC. Twenty-five different complotypes were identified, among which there were four complotypes that had not been previously observed in our large database of complotypes compiled from family studies of Boston Caucasians and that are, presumably, unique to individuals of African origin. These four African-derived complotypes are FC(1,90)0, FC63, S1C2, 17, and SC(3,2,90)0. The frequencies of two of these four unique Black complotypes, FC(1,90)0 and FC63, were increased significantly when compared to Caucasians (pcorr less than 0.00042, pcorr = 0.00294, respectively). The complotype FC(1,90)0 was in positive linkage disequilibrium with HLA-DR3 haplotypes containing the B locus antigens Bw42, Bw52, Bw53, and Bw58, while FC63 was associated with HLA-Bw70, -DR5. These findings demonstrate the extensive polymorphism of complotypes in Blacks, and also suggest that it may be possible to define unique extended haplotypes of African origin.

Structural model for T-cell recognition of HLA class II—associated alloepitopes

In an effort to investigate the structure-function relationship of HLA class II molecules vis-à-vis alloepitope expression, cloned T-cell reagents were used to define polymorphic epitopes associated with DR and DQ molecules. DNA sequences of genes encoding allelic or isotypic DR or DQ molecules that appear to express the same T-cell-defined epitopes were compared in an attempt to identify association of shared sequences with shared epitopes. When sequence sharing is associated with shared epitope expression, we suggest that it is the shared sequence that encodes the epitope in question. Based on the hypothetical three-dimensional structure of the class II molecule, an approximation is made as to which parts of the HLA class II molecule are involved in alloepitope expression. T-cell clones were generated from cells primed against HLA-DR2 haplotypes representing the cellularly defined subgroups Dw2 or Dw21 (previously designated MN2, FJO, or Tb24). Those clones determined to be DR- or DQ-directed based on monoclonal antibody inhibition assays were tested by panel cell analysis utilizing DR2-positive and DR2-negative target cells. The data support the concept that amino acids 67, 70, 71, and 74 for DR molecules and amino acids 57, 70, and 71 for DQ molecules, which appear to comprise one face of the α helix, are of primary importance in T-cell recognition. In other cases, sharing of both the second hypervariable region (amino acids 25–33) and the third hypervariable region (amino acids 67–74) appears necessary to explain epitope sharing for DR molecules. We emphasize that the involvement of these two hypervariable regions may indicate that alloepitope expression involves the complex of class II molecule plus peptide, with the second HVR primarily involved in determining which peptides are bound and the third in T-cell receptor (TcR) recognition and/or peptide binding; we do not rule out that conformational changes of the second HVR can induce conformational changes in the third HVR. Finally, shared alloepitopes detected by some clones could not be explained based on shared primary sequences.

FAMILIAL PATTERNS OF NARCOLEPSY

Familial patterns of narcolepsy were investigated in a clinic population of 334 unrelated narcoleptic patients. 40% of probands had at least 1 family member with an isolated daytime sleepiness complaint and 6% had a positive family history of narcolepsy. Multicase families were rare; only two families were found with 3 or more affected relatives. Family members often shared the same HLA-DR2 haplotype as the proband but did not have narcolepsy. However, the risk of disease for first-degree relatives was six to eighteen times greater than that for unrelated individuals. Although most patients were HLA-DR2+, 2 new HLA-DR2- individuals were found. The data predict that as many as 9% of unrelated North-American white patients with narcolepsy will be DR2-. Analysis of these and other data indicates that although strongly associated with disease, the HLA-DR2 haplotype is neither sufficient nor necessary for the development of narcolepsy.

Normal in vitro production of TNF in healthy subjects with HLA-DR2 haplotype and in patients with narcolepsy

Normal in vitro production of TNF in healthy subjects with HLA-DR2 haplotype and in patients with narcolepsy

Structural analysis of the HLA-DR, -DQ, and -DP alleles on the celiac disease-associated HLA-DR3 (DRw17) haplotype.

Celiac disease is strongly associated with the HLA class II D-region serologic markers DR3 (DRw17) and DQw2. Moreover, by restriction fragment length polymorphism analysis, greater than 90% of DR3 (DRw17), DQw2 celiac disease patients have a polymorphic 4.0-kilobase Rsa I DP B gene DNA fragment. The present study sought to determine if there is a unique HLA class II D-region A or B gene structural variant on the DR3 (DRw17) haplotype found in celiac disease. The polymorphic second exons of the coding DRB, DQA and DQB, and DPA and DPB genes in celiac disease patients with the DR3 (DRw17) haplotype were sequenced after amplification by the polymerase chain reaction. To define the DP B genes associated with celiac disease, the second exons of the coding DP B genes from 27 celiac disease patients were amplified similarly and probed by using a panel of sequence specific oligonucleotides. The HLA-DR, -DQ, and -DP A and B gene second exon sequences of celiac disease patients were noted to be identical to sequences that can be found also, although at a significantly lower frequency, in unaffected individuals. This is compatible with a disease model wherein the HLA class II genes on the DR3 (DRw17) haplotype are necessary, but not sufficient, for the phenotypic expression of celiac disease. Analysis of the DP B genes revealed a significant increase in the frequency of the alleles DPB1 and DPB3 in celiac disease. Furthermore, the increased frequency of the 4.0-kilobase Rsa I DP B gene restriction fragment length polymorphism in celiac disease can be accounted for by the overrepresentation in disease of the alleles DPB1 and DPB3. The HLA-associated susceptibility to celiac disease appears to be multigenic, with specific, but structurally normal, allelic variants in the DP and DQ/DR subregions contributing to disease susceptibility.

DIDMOAD Syndrome and HLA-DR Haplotypes

DIDMOAD Syndrome and HLA-DR Haplotypes

HLA-DR and tuberculin tests in rheumatoid arthritis and tuberculosis.

Responses to four new tuberculins were found to be significantly reduced in 46 patients with rheumatoid arthritis in comparison with a control group of 79. Except for tuberculin itself, the same was found in 111 patients with tuberculosis. In common with patients with tuberculosis and leprosy, those with rheumatoid arthritis did not respond to common mycobacterial (group i) antigen. Three DR haplotypes were found to have significant effects on skin test responsiveness of the rheumatoid patients but had little or no effect on that of the patients with tuberculosis and none on that of the healthy control group. Rheumatoid patients with the HLA-DR4 haplotype had significantly greater responses to all four reagents than did non-DR4 patients, but their responses to leprosin A and scrofulin remained significantly lower than those of the control group. Possession of HLA-DR3 haplotype was associated with skin test positivity approaching normal, but the sizes of responses were reduced. Possession of DR7 was associated with an unexpected reduction in skin test positivity, especially in the case of tuberculin. These results support the hypothesis that mycobacteria, or autoantigens cross reactive with mycobacteria, may be involved in the aetiology of rheumatoid arthritis. The results also show that the regulation and specificity of responsiveness to mycobacterial antigens are different in patients with rheumatoid arthritis with different HLA-DR haplotypes.

Two distinct HLA-DR3 haplotypes are associated with age related heterogeneity in type 1 (insulin-dependent) diabetes.

Heterogeneity between two haplotypes in linkage disequilibrium with DR3: B8, C4AQOB1,BfS,DR3 and B18,C4A3BQO,BfF1,DR3, with regard to age at onset of Type 1 (insulin-dependent) diabetes mellitus, was investigated in 325 unrelated French patients (146 males and 179 females, age at onset 1 month to 29 years) who were genotyped for HLA-A, B, C, DR and Bf and 225 of whom were typed for the C4A, B complement components. A subgroup of 82 patients and 75 control subjects were tested for DR beta and DQ beta DNA restriction fragment length polymorphism. The distribution according to age at onset and the mean ages at onset were compared between patients bearing B8, DR3 (n = 58), B18,DR3 (n = 62) or other DR3 haplotypes (Bx, DR3, n = 70), the haplotype segments C4AQOB1,DR3 (n = 41) or C4A3BQO,DR3 (n = 52) and the C4 null alleles C4AQO (N = 48) or C4BQO (n = 112) alone. The B8,DR3 haplotype, its smaller segment C4AQOB1,DR3 or C4AQO alone were associated with age at onset after 6 years (p less than 0.01, less than 0.08 and less than 0.02 respectively); on the other hand, the B18,DR3 haplotype, its segment C4A3BQO,DR3 or C4BQO alone were significantly more frequent in patients aged less than 6 years at onset (p less than 0.02, less than 0.01 and less than 0.01 respectively). Accordingly, the mean age of onset was significantly lower in the latter compared with the former patients (p less than 0.02, less than 0.02 and less than 0.01 respectively). No age-related variation was observed in BX,DR3 patients and their mean age of onset was intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)

HLA-Dw14 and HLA-DR3 haplotypes share a functional determinant recognized by a human alloreactive T-cell clone

In the process of studying the fine specificity of HLA class II molecules, we identified an alloreactive T-cell clone raised to a HLA-Dw14 homozygous cell line that was specifically stimulated by Dw14+ homozygous typing cells but negatively with cells expressing the HLA-Dw4, -Dw10, -Dw13, and -Dw15 subspecificities of DR4. Of interest, this clone was also equivalently activated by stimulation with all DR3 cells and cell lines tested. Negative responses were obtained using a panel of 87 non-DR3 and non-Dw14 cells, including cell lines of the Tenth Histocompatibility Workshop. A monoclonal antibody inhibition study revealed the relevant stimulating determinant to be on HLA-DR molecules in both Dw14- and DR3-positive cells. A comparison of the DRβ1-chain-inferred amino acid sequences suggests that formation of a topologically equivalent stimulating determinant would involve the participation of two noncontiguous regions of the third diversity region of DRβ1. The putative recognition conformation detected by the clone is most probably specified by the presence of a valine at position 86 and a nonnegatively charged residue at positions 70, 71, and 74, since these are the only residues where DR3 and Dw14 are distinguishable from all other HLA-DR types. These findings illustrate that the functional ability of class II molecules is not necessarily either illustrated or predicted by serologic typing or by simple consideration of amino acid sequence.

HLA-DQw Alloantigens and Pulmonary Dysfunction in Rheumatoid Arthritis

HLA-DR4 and keratoconjunctivitis sicca (secondary Sjögren's syndrome) are associated with abnormal pulmonary function in patients with rheumatoid arthritis. Since recent investigations have found that much of the genomic polymorphism of the HLA-DR4 haplotype comes from the closely linked DQw allele, we reanalyzed this set of data to evaluate the relationship between the DQw allotypes and pulmonary function in rheumatoid arthritis. Using a step-wise regression analysis, we found that the presence of DQw1 was a stronger predictor of an abnormal forced vital capacity (FVC), forced expiratory volume in one second (FEV1), and carbon monoxide diffusing capacity (D) than the presence of DR4, keratoconjunctivitis sicca, smoking status, or any other clinical parameter. DQw1-positive patients had a mean (+/- SD) percent of predicted FEV1, FVC, and D of 84.2 (+/- 19.8), 88.0 (+/- 17.9) and 85.6 (+/- 20.9) percent, respectively, all significantly lower than DQw-1 negative patients (p = 0.02, 0.02, and 0.03). Smokers with the heterozygous phenotype, DQw1/DQw3, tended to have obstructive disease of the airways, with a mean (+/- SD) FEV1 of 80.1 +/- 24.4 percent of predicted, compared to 95.7 +/- 12.1 percent of predicted in DQw1/DQw3-negative individuals (p = 0.03). Patients who had a DQw2 allele were more likely to have normal pulmonary function. We conclude that the HLA-DQw1 allotype is a strong predictor of abnormal pulmonary function and that it may identify smoking subjects with rheumatoid arthritis subjects who are prone to develop obstruction of airflow.

Hybridization analysis reveals novel DQw3 alpha-beta associations suggesting a recombination point between DQ alpha and DQ beta on the HLA-DR4 haplotype.

Hybridization analysis reveals novel DQw3 alpha-beta associations suggesting a recombination point between DQ alpha and DQ beta on the HLA-DR4 haplotype.

The influence of culture conditions and serum lipids on interleukin-1 production by human monocytes

Reliable assessment of IL-1 production by human monocytes is critically dependent on the methods for isolation and culture of these cells. In the present study, the quality of pipettes and the preparation of Ficoll-Isopaque appear to be crucial for IL-1 production from both LPS-stimulated and unstimulated monocytes. Different brands and lots of polystyrene culture wells give rise to great variation in IL-1 production. When carefully prepared, hydrophobic teflon membranes, to which mononuclear phagocytes poorly adhere, are used as the culture substrate, stimulation of IL-1 production is observed. The HLA DR3 haplotype of the monocyte donors did not influence IL-1 production. The addition of normal human AB serum to the cultures usually increases IL-1 production, although strong inhibition of both unstimulated and LPS-stimulated IL-1 production was also observed after addition of a diet-induced hyperlipemic AB serum. This inhibition was not due to cholesterol, chylomicrons, high- or low-density lipoproteins. When monocytes were cultured at different temperatures, the only abnormality found was a decrease of cell-associated IL-1 at 41°C.

Narcolepsy-cataplexy in Israeli Jews is associated exclusively with the HLA DR2 haplotype A study at the serological and genomic level

Narcolepsy is a very rare disease among Israeli Jews with a frequency of 713 × 10 6. As investigation of the association of narcolepsy with the human leukocyte antigen system was conducted in Israeli Jews at the serologic and genomic levels. The human leukocyte antigen class I and class II antigen typing of 7 clinically diagnosed narcoleptics, 3 individuals suffering from sleep disorders other than narcolepsy, and 11 healthy matched controls revealed that all narcoleptic patients (100%) investigated in the present study carried the HLA-DR2 haplotype, whereas patients with other sleep disorders did not. The HLA-B7 and DR2 occurred jointly in 57% (4/7) of the narcoleptic patients, as compared to 2% in randomly selected Israeli healthy controls. Restriction fragment length polymorphism analysis was performed with several restriction enzymes and three cDNA probes for DQα, DQβ, and DRβ genes on genomic DNAs obtained from narcoleptics and patients with other sleep disorders, matched controls, and 3 homozygous typing cells representing the DR2 subtypes Dw2, Dw12, and DwAZH. The restriction fragment length polymorphism analysis showed that all narcoleptics (7 of 7) shared virtually identical restriction fragment length polymorphisms with one of the homozygous typing cells (GSO), which defines DR2, Dw2. The frequency of the DR2,Dw2 haplotype in the healthy Israeli population is 3.2%. Other non-narcoleptic patients did not share these restriction fragment length polymorphisms. These findings indicate that narcolepsy is associated worldwide with the HLA-DR2,Dw2 haplotype.

HLA-DQ BETA-CHAIN POLYMORPHISM IN HLA-DR4 HAPLOTYPES ASSOCIATED WITH RHEUMATOID ARTHRITIS

With a cDNA probe for the DQβ gene, two variants of the DR4-linked DQw3 (3.1 and 3.2) allele were analysed in patients with rheumatoid arthritis (RA), healthy individuals, and homozygous cell lines. The DQw3.1 allele, identified by 3.4 kb ( HindIII), 2.3 kb ( SstI), and 3.7 kb and 6.9 kb ( BamHI) restriction fragment lengths, was expressed in 100% (of 18) DR4-positive patients, compared with only 19% (of 16) of DR4-positive controls including one of five homozygous cell lines. This DQβ variant showed a highly significant association (relative risk = 78; p < 10 -6) with RA and may therefore play an important part in susceptibility to RA.

T-cell clonal analysis of HLA-DR2 haplotypes

Individuals carrying the serologically-defined DR2 haplotypes can be further subdivided by utilizing T cell response to define several HLA-Dw/LD clusters: Dw2, Dw12, LD-5a, and a related group composed of FJO, AZH, and MN2. We have used cytotoxic T cell clones generated against these DR2 haplotypes to define the DR and DQ associated Dw/LD polymorphisms. Clones were categorized as DR, DQ, DP, or class 1-directed based on monoclonal antibody (MoAb ∗) inhibition studies. We analyzed the distribution of the determinants detected by these clones based on analysis with panels of cells. Some DR-directed clones only lysed cells positive for the Dw subtype of the sensitizing cell. A few DR-directed clones lysed most DR2 positive cells. Other DR-directed clones detected determinants shared by cells of different subtypes: determinants shared by Dw2 and Dw12 positive cells and determinants shared by MN2, FJO, AZH, and LD-5a positive cells. The majority of the DQ-directed clones only lysed cells positive for the sensitizing HLA-Dw subtype. A group of clones primed against an LD-5a HTC, whose cytolytic activity was not blocked by any monoclonal antibodies tested, lysed some, but not all, DR2 positive cells as well as Dw1 and Dw10 positive cells. The results suggest the detection of determinants on these molecules which may be shared by cells of specific Dw subgroups and determinants shared by most cells of the sensitizing serological specificity. The basis of this observed polymorphism and influence on the allogeneic response are discussed.

Microheterogeneity of HLA-DR4 haplotypes: DNA sequence analysis of LD“KT2” and LD“TAS” haplotypes

We have isolated and sequenced cDNA clones corresponding to the polymorphic α and β chains encoded by the DR and DQ subregions of two HLA-DR4 haplotypes, LD“KT2” and LD“TAS”. These two haplotypes are distinguished on the basis of mixed lymphocyte culture typing. The data indicate that the designation of LD“TAS” as a distinct subtype from Dw13 is very likely due to amino acid differences in the DQβ chain. In contrast, LD“KT2” differs from TAS and Dw13 by a single amino acid substitution at position 37 of the DRβ1 chain. The functional and evolutionary significance of these polymorphisms is discussed.