An overview of the restriction fragment length polymorphism of the HLA-D region: its application to individual D-, DR- typing by computerized analyses

HLA-D/DR alleles as defined by cellular and serological typing can also be identified by biochemical methods. The Southern blot technique provides an additional typing facility which can be applied to DNA obtained from any source of nucleated cells. The polymorphism revealed by Southern blot analyses, the so-called restriction fragment length polymorphism (RFLP), depends upon the restriction enzyme and cDNA probes used. To identify HLA-DR specificities a protocol was developed based on the use of the results of southern blot analyses with several restriction enzymes and cDNA probes within a panel of HLA-D/DR homozygous cells representing the DR1 to DRw8 alleles. First, hybridizations with the 3' untranslated sequence of the DR beta cDNA probe, after digestion of the DNA with PvuII (PvuII-DR beta 3') allows the selective identification of DR1, DR2 and DRw8; DR3, DR5 and DRw6 are found as one group as well as DR4 and DR7 as another. Second, TaqI-DQ alpha hybridization allows the splitting of DRw6-Dw18, DRw6-Dw19 and DRw6-Dw9 from the DR3, DR5 and DRw6 group. The other alleles DR3, DR4, DR5, DRw6-Dw16 and DR7 are revealed by dehybridization and rehybridization of the blot with a DR beta cDNA probe. This protocol was used to test whether in a panel of 30 randomly chosen individuals the HLA-DR typing could be performed. The results were highly concordant to the serotyping. Furthermore by adding the Pst-DR beta and TaqI-DQ alpha RFLPs, most of the MLC defined Dw specificities could also be identified. An overview of the specific fragments described here has been summarized in matrices which can be used as references for DNA-typing in computerized analyses.     

Oligonucleotide-genotyping as a method of detecting the HLA-DR2 (DRw15)-Dw2, -DR2 (DRw15)-Dw12, -DR4-Dw15, and -DR4-D"KT2" haplotypes in the Japanese population

We synthesized pairs of four different oligonucleotides, F22, F29, F42, and F158, to analyse the HLA-DR2 (DRw15) and -DR4 haplotypes in the Japanese population. After enzymatically amplifying the HLA-DRB1 gene, we hybridized the oligonucleotide probes with DNA extracted from 42 donors. Hybridization was completed between F22 and the DNA of haplotype DR2 (DRw15)-Dw2, between F29 and the DNA of DR2 (DRw15)-Dw12, between F42 and the DNA of DR4-D"KT2", and between F158 and the DNA of DR4-Dw15. In keeping with the nucleotide sequences of the probes, F29 hybridized also with DNA from the DR9-Dw23 haplotype and F158 with that from some of the DRw8 haplotypes (DRw8-Dw8.3) in the Japanese population. Results of this study demonstrate that the four oligonucleotides make useful probes for detecting the haplotypes above.     

The origin of HLA-DR"Br": exon 2 nucleotide sequence implicates possible gene conversion of DR1 by DR4-Dw10, DR5, or DRw6-Dw18

The exon 2 nucleotide sequences of HLA-DQwl-associated and DQw3-associated HLA-DR"Br" alleles were determined from genomic DNA amplified by the Taq polymerase chain reaction technique. Both alleles reveal identical exon 2 nucleotide sequences. Comparison with other DR alleles suggests that DR"Br" may have originated from DR1 by gene conversion with DR4-Dw10, DR5, or DRw6-Dw18 third hypervariable region sequences.     

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 DR beta 1-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 DR beta 1. 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 considerations of amino acid sequence.     

Serology, restriction fragment length polymorphism, and sequence analysis of a unique HLA class II antigen, DR5x6.

We analyzed a new class II HLA haplotype, which we have designated DR5x6, by serology, restriction fragment length polymorphism (RFLP), and sequence analysis. As the name DR5x6 implies, the antigen is serologically closely related to both DR5 and DRw6. RFLP analysis of this haplotype suggests a close similarity with DRw11 haplotypes. The DNA sequences encoded by the second exon of its DRB1, DRB3, and DQB1 genes were also determined. Comparison of these sequences with those of alleles at these loci in other haplotypes suggests that this haplotype could have evolved from a DRw11 ancestor haplotype (DRw11-DRw52b (Dw25)-DQw7) by means of: (a) a gene conversion at the DRB1 locus involving DRw8 (Dw8.3) as the sequence donor, plus a point mutation or a gene conversion involving DR4-Dw4; and (b) a recombination event by which this haplotype would have acquired the DRw5a (Dw24) allele at the DRB3 locus.     

Typing for HLA-D Determinants. Comparison of Typing Results using Homozygous Stimulating Cells and Primed Cultures

Typing for HLA-D determinants, using primed lymphocyte typing (PLT), has been compared to the results obtained using conventional homozygous stimulating cell primary MLC tests. The HLA-Dwl, -Dw2 and -Dw3 specificities were studied. Preliminary results indicate that these two methods essentially type for the HLA-D determinants and that reproducible results can be obtained employing the PLT technique after 24 hours of incubation in the secondary cultures.     

Combined use of RFLP and PCR-ASO typing for HLA-DR-Dw and DQw typing.

Due to some limitations of restriction fragment length polymorphism (RFLP) analysis in HLA-DR-DQ typing, we present a combined use of RFLP and polymerase chain reaction (PCR)-allele-specific oligonucleotide (ASO) typing. This scheme consists in selectively amplifying the few RFLP ill-defined genes (DR1/DR'Br' and DR4-Dw subsets) using PCR with allele specific primers to avoid cross-hybridization.     

Class-II HLA Restriction of Antigen-Specific Human T-Lymphocyte Clones

Blast-enriched suspensions of T cells primed for Chlamydia trachomatis antigen were cloned by a limiting dilution technique. The class-II HLA restriction of T-lymphocyte clones (TLC) was studied by using allogeneic antigen-presenting cells (APC) carrying foreign class-II HLA antigens. Most of the TLC were restricted by one or the other of the D/DR determinants of the T-cell donor; that is, they did not respond when antigen was presented by APC expressing foreign D/DR determinants. Furthermore, heterogeneity of the DR4-expressing molecule could be demonstrated by T-cell clones from one person; APC from family members expressing DR4 gave high proliferative responses, whereas no proliferation was observed with most APC from unrelated persons expressing DR4. This heterogeneity of DR4 was confirmed by mixed lymphocyte culture (MLC) experiments, indicating a close relationship between restriction epitopes and those that activate allogeneic T cells. Other clones seemed to be restricted by other class-II HLA determinants, most probably MT determinants of the T-cell donor. The restriction specificities were confirmed by subcloning experiments.     

DQ β sequences in HLA-DR4 haplotypes

A cDNA clone encoding the entire mature DQ β chain was isolated and sequenced from the DR4-Dw14 homozygous cell line, LS40. The sequence was compared with published DQ β sequences from cells expressing DR4-DQw3, and found to be identical. The lack of DQ β sequence polymorphism within this serotype (to date, DQ β sequences have been derived from five cell lines comprising three Dw subtypes) adds to the data that suggest a recent evolutionary divergence of Dw subtypes within DR4-DQw3.     

Molecular characterization of a recombinant HLA-DR1/DR2 haplotype

Serologic analysis of two families identified an HLA-DR haplotype in which DR1 and DR2 cosegregated. DNA-RFLP analysis of these families with an HLA-DRB probe revealed a pattern of hybridization suggestive of a recombination between DR1 and DR15. Following amplification, cloning, and nucleotide sequencing of HLA-DRB-gene second-exon DNA sequences, three DRB amplification products associated with the novel haplotype were identified: these corresponded to DRB1*0101, DR2 pseudogene, and DRB5*0101. Clones representing the DRB1*1501 and DR1 pseudogenes were not identified: oligonucleotide typing with DRB1*1501-specific probes confirmed the absence of this gene within the DR1/DR2 haplotype. We postulate that the DR1/DR2 haplotype represents a recombinant between those of DR1-Dw1 and DR15-Dw2, and that the crossing-over may have been between the DRB1*0101 gene and the DR2 pseudogene. This is further supported by DNA-RFLP analysis with HLA-DQB and DQA CDNA probes, which revealed conserved linkage genes between the DQB1*0501, DQA1*0101, and DRB1*0101 genes.     

DNA typing for class II HLA antigens with allele-specific or group-specific amplification. IV. Typing for alleles of the HLA-DR2 group

In this study we present an approach for the definition of the alleles belonging to the HLA-DR2 group by DNA typing with oligonucleotide probes. Following methodology similar to that we used previously for the definition of other HLA-DR subsets, we have now developed primers for DR2-DRB1 and DR2-DRB5 amplification, and probes for the identification of sequences that distinguish the subtypes of this group of genes. The method used defines all the previously described alleles at both DR2-associated DRB loci. In addition, we have identified a variant of DRB1-DR2-Dw2. This new allele has been called DRB1*15.3. It is different from DRB1*1501 in codon 30, where it carries histidine instead of tyrosine. Eight different haplotype combinations of DRB5, DRB1, and DQB1 were identified within the DR2 group and their occurrence in four normal panels of different ethnic origin has been described. Haplotypes containing DRB1*15.3 occurred most frequently in black panel members in whom it was associated with either DQB1*0602 or DQB1*0501. Two unusual haplotypes were observed: one containing elements of DR2-Dw21 (DQB1*0502) and of DR2-Dw22 (DRB1*1602) and one containing elements of Dw21 (DRB1*1601, DQB1*0502) and Dw2 (DRB5*0101). The methods described permit simple and rapid determination of the alleles of the HLA-DR2 group and should be useful for population studies and for investigation of DR2-associated diseases.     

Techniques used to define human MHC antigens: restriction fragment length polymorphisms.

Polymorphisms within the HLA-DRB1, -DRB3, -DQB1 and -DQ A1 genes are detectable using restriction fragment length polymorphism (RFLP) analysis. DNA is isolated from EDTA-treated blood or from spleen or lymph nodes. The DNA is digested to completion with the restriction endonuclease TaqI and resolved using agarose gel electrophoresis. The DNA after denaturation is then transferred to a nylon membrane (Southern blotting) and hybridised with radiolabelled cDNA probes: HLA-DR beta pRTV1, HLA-DQ beta pII-beta-1 and HLA-DQ alpha pDCH1. After autoradiography the membrane is dehybridised prior to rehybridisation. This system is very useful in those situations where serological assignment is difficult due to poor quality or low numbers of circulating B cells and where there is a lack of reliable antisera for certain specificities. The RFLP techniques can also define subtypes of DR and DQ serological specificities. However, certain alleles have the same RFLP. In some instances by identifying the DQ allele the DR allele can be determined by association due to linkage disequilibrium (e.g., DRw17-Dw25-DQw2 and DRw13-Dw25-DQw6). In other instances (e.g., DR1 and DRBr), the problem can be resolved using serology. In addition the RFLP system cannot be applied prospectively to the cadaver donor situation because of time restrictions. Thus the RFLP system complements existing serological techniques. However, it can be very useful as a quality control for the serological methods especially in the assessment of the quality of antisera and in the determination of discrepancies between centres.(ABSTRACT TRUNCATED AT 250 WORDS)     

HLA-DR characterization of a Chippewa Indian subpopulation with high prevalence of rheumatoid arthritis

The adult population (N = 227) of a Chippewa Indian reservation in central Minnesota is characterized by a high prevalence of arthropathy with 7.1% having rheumatoid arthritis (RA). In a prospective study 168 reservation residents (74%) were evaluated. Complete HLA typing identified 57 haplotypes, many of which probably arose via HLA-A/B or B/D recombination. The number of founder haplotypes appeared to be about 20. The population frequency of DR4 (including DRw9, formerly designated 4 X 7) was 67%; for RA it was 100% (p less than 0.05). Apparent DR4 homozygotes, a number of the RA patients, and family members were selected for further study in a mixed lymphocyte culture (MLC) test and with 8th International Workshop cellular and serologic reagents. In MLC with homozygous typing cells (HTC), non of the DR4+ cells typed for any known HLA-D specificity, although they reacted to all DR4 antisera on the local panel. However, 8th International Workshop DR antisera revealed patterns of reactivity with non-DRf4 reagents consistent with the MLC. A minimum of three DR4 variants, one DRw9 variant, and a specificity related to both DR4 and DR2 are required to explain both the cellular and serologic reactions. For the present, we are designating the antigens as DR4.1chip, DR4.2chip, DR4.3chip, DR9chip, and DR(2 X 4)chip. No single variant of DR4 was characteristic of the RA patients in this Amer-indian population.     

HLA-DQA1 and MLC among HLA (generic)-identical unrelated individuals

We modified a previously published PCR-RFLP for DQA1 typing (1) and examined the predictive value of HLA-DQA1 in mixed lymphocyte cultures (MLC) among matched (HLA generic types) pairs of unrelated individuals. There were 61/102 (60%) pairs with positive MLC, one-third of which could be predicted by DQA1* typing alone. DQA1 matching and MLC reactions were classified into 3 groups: 1) DQA1 mismatches showing positive MLC: 19/102 (19%); 2) DQA1 matches showing negative MLC: 41/102 (40%); 3) DQA1 identical showing positive MLC: 42/102 (41%). Five different HLA haplotypes that result from non-random association of HLA generic types (high delta haplotypes) were overrepresented in the individuals tested. One of these haplotypes carrying HLA-B7, DR2 was found associated with three different DQA1 alleles (*0201, *0103, *0102). The remaining four high delta haplotypes were associated with one DQA1 allele in all independent examples tested: HLA-A1, B8, DR3 with DQA1*0501; HLA-A26, B38, DR4 with DQA1*0301; HLA-A2, Bw62, DR4 with DQA1*0301 and HLA-A1, Bw57, DR7 with DQA1*0201. Forty per cent of the negative MLC were explained in part by the excessive number of individuals carrying two of these four haplotypes, which probably carry determinants in linkage disequilibrium with HLA. Nineteen per cent of HLA-identical (generic types) unrelated pairs show positive MLC reactions and all of them are DQA1* mismatched, suggesting that DQA1* allele typing should be used to screen samples prior to performing MLC.     

DR2+ haplotypes in insulin-dependent diabetes: analysis of DNA restriction fragment length polymorphisms

Insulin-dependent diabetes (IDD) is strongly associated with certain HLA class II (Ia) antigens. The frequency of DR2 is significantly reduced in IDD; among DR2+ patients, the frequency of the subtype specificity Dw2 defined with homozygous typing cells (HTCs) is significantly reduced compared to DR2+ controls, and the specificity LD-MN2, which we have defined using primed lymphocyte typing reagents, is significantly increased. We have studied DNA restriction fragment length polymorphisms (RFLP) of DR2-LD-MN2+ individuals and homozygous typing cells carrying specificities antigenically related to LD-MN2. Using a number of different restriction enzymes, a characteristic pattern of fragments could be defined for DR2-LD-MN2 using both DQ beta and DR beta cDNA probes. This pattern was shared with some but not all of the antigenically related HTCs, and was distinct from that of DR2-Dw2. The RFLP pattern of DR2-LD-MN2 obtained with the DQ beta probe is identical, except for one band, to that of DR1-Dw1, suggesting that at least some part of the DQ region is identical in these two haplotypes. These results indicate that analysis of RFLP patterns can be used to help identify the genetic regions and, eventually, genes most important in the association of HLA and IDD.     

HLA antigen associations with extra-articular rheumatoid arthritis

Seventy-seven patients with rheumatoid arthritis were investigated to examine the frequency of HLA antigens and their relationship to clinical and serological manifestations of extra-articular disease. The phenotype frequencies of DR4, DRw53, Bw62 and Cw3 were significantly increased, compared to normal controls, and there were negative associations with DR2 and DR7. The HLA antigen in strongest association with rheumatoid arthritis was DR4 (73.6%) and the relationship with DRw53 appeared to be secondary. The frequency of DR4 rose to 92% in seropositive patients with extra-articular disease manifestations whose serum contained immune complexes. A high frequency of DR4 was also seen in male patients (86%), reaching 100% in the small group of seropositive male patients with immune complexes. It is suggested that extra-articular disease represents a manifestation of severe classical rheumatoid arthritis and is not an 'overlap' syndrome. We propose that the HLA haplotype Cw3-Bw62-Dw4-DR4-DRw53 makes a greater genetic contribution to disease susceptibility in both extra-articular and male rheumatoid arthritis patients than in other subsets of RA.     

上海人群中HLA-DR7单倍型同时表达两种纯合分型细胞检出的特异性Dw7c及Dw3

采用国际组织相容性会议提供的纯合分型细胞(HTC)和血清对上海地区56例无亲缘关系个体作HLA-A、B、C、D、DR、DQ分型并研究中国人DR-Dw关系后,发现11例Dw3阳性个体中仅5例表达命名相当的DR3特异性,另外6例Dw3阳性者却与DR7及Dw7c(Dw7+Dw17)共同表达于一条单倍型,使同一个体呈现HLA-D“三联体”这样一种未曾报导过的格局。中国人Dw3因而分成两类:一类见于传统的单倍型HLA-DR3-Dw3;另一类组成新单倍型HLA-DR7-Dw7c-Dw30。间接证据表明,后者的出现可能是中国人中一个新的HLA-DQw2分裂体同时参与Dw7c及Dw3功能表达并被HTC所识别的结果。     

Recognition of DP determinants with typing reagents prepared with lymphocytes from Dutch unrelated individuals

In an attempt to make our own set of DP typing reagents, we used lymphocytes from 12 unrelated donors, who were all HLA-A1,2; B7,8; DR2,3; DQW1,2. They all had been previously typed for DP using a reference set of well established reagents obtained from Dr. S. Shaw (NIH, Bethesda). Thirty-six promising responder-stimulator combinations were primed in bulk MLC and tested for their specificity in secondary MLC. All reagents gave reaction patterns which were concordant with the sensitizing DP types, with the exception of those combinations where a donor was used in which DR2 appeared to be associated with a non-DW2 HLA-D type. Over 1,200 reactions obtained with the new reagents were compared with those obtained with the established ones, in six different experiments. High correlation coefficients (r values) were found between the two kinds of reagents. The typings of a panel of individuals with the reference set and with our new typing set revealed an excellent agreement for DP assignments with the two sets, with the exception of the specificity DP4. The DP gene frequencies for random Dutch Caucasoids were defined.     

A monoclonal antibody detecting an HLA-DQwl-related determinant

A complement fixing monoclonal antibody (moab) was prepared which reacts with a polymorphic determinant on HLA class II molecules. The moab IIB3 recognises all DQwl (DC1, MB1, LB-E12) positive cells as well as some DR4, DR7, DRw8 and DRw9 positive cells. The moab reacts mainly with B-cells and not or with only a minority of the monocytes. Segregation of the determinant with HLA-DR could be shown. The determinant is strongly expressed on DR2, DR4 and DRw6 positive cell lines but only weakly on DR1 lines. In contrast to a monoclonal antibody against a monomorphic determinant on class II molecules IIB3 did not give a distinct inhibition of the MLC nor did it inhibit the generation of CTLs in MLC as has been described for the DQwl like moab BT 3/4 (Corte et al. 1982). Immunoprecipitation indicates that IIB3 reacts with DQ-like molecules.     

Heterogeneity of HLA-D(DR)4 detected by primary and secondary mixed-lymphocyte-culture reactions and by individual HLA-DR4 antisera

One of our panel families (Sb), in which the paternal haplotypes Dw4, DR4 (a) and Dwblank, DR4 (b) segregate, was tested in primary mixed lymphocyte culture (1.MLC) and in the primed lymphocyte test (PLT). In the 1.MLC, cells which carry the a haplotype strongly stimulate b-haplotype cells, and vice versa. For the PLT, lymphocytes of two family-members were primed against the a haplotype and two against the b haplotype. A strong positive restimulation (RR greater than or equal to 60%) occurred only with cells bearing the original stimulating haplotype. The PLs were tested later against families St and Sm, which possess DR4 haplotypes, and against a panel of 73 unrelated persons. The results show heterogeneity of D(DR)4, suggesting at least three different subgroups: D(DR)4a, present on DR4 cells which strongly restimulate the anti-a PLs; D(DR)4b, on DR4 cells which strongly restimulate the anti-b PLs, and D(DR)4c, on the DR4 cells, which do not restimulate any of the PLs tested here. It seems also possible to differentiate between these subgroups with conventional DR-serology, as the 8W sera 903 and 981 react only with a-haplotype cells of family Sb, and ths 8W sera 872 and 1045 react only with b-haplotype cells.