Peter T. Klouda

A DNA-RFLP typing system that positively identifies serologically well-defined and ill-defined HLA-DR and DQ alleles, including DRw10.

A single enzyme/multiple probe system of HLA-DR and DQ typing using restriction fragment—length polymorphism (RFLP) analysis is presented. TaqI-digested genomic DNAs are hybridized sequentially with short DRβ, DQβ, and DQα cDNA probes. The DRβ probe discriminates between the DR allelic specificities DR1 to DRwl4, with the two exceptions of some DR3/DRwl3 and some DR7/DRw9 combinations. We describe the positive identification of a DRw10-specific RFLP and demonstrate its segregation in families. The DQβ probe defines an allelic system that identifies the alleles DQw1, DQw2, and DQw3. This permits the resolution of DR3/DRw13 and DR7/DRw9 alleles by defining the DR/DQ association caused by linkage disequilibrium. The DQα probe defines another allelic series interrelated with, but independent from, the DQβ series. Specific DQβ/DQα RFLP combinations correlate with known Dw splits of DR2, DRw6, and DR7. Combined use of the three probes permits the identification of HLA-DR, DQ, and certain Dw specificities and provides an effective and easily interpretable system for major histocompatibility complex class II allogenotyping.

Allogenotypes defined by short DQα and DQβ cDNA probes correlate with and define splits of HLA-DQ serological specificities

Abstract HLA-DR and -DQ serotyped cell lines and peripheral blood leucocytes were analysed by Southern blot allogenotyping. Using a short DQβ cDNA probe, a DQβ allelic series was defined by restriction fragment length polymorphism (RFLP) with the restriction endonuclease Taq I. This DQβ allelic series correlates with, and defines splits of, the HLA-DQ serological specificities DQw1 (DQβ1a and DQβ 1b RFLPs), DQw2 (DQβ2a and DQβ2b RFLPs) and DQw3 (DQβ3a and DQβ3b RFLPs). By sequential use of a short DQa cDNA probe a second, DQα allelic series is defined by RFLP. This series correlates to a lesser extent than DQβ RFLPs with the HLA-DQ serological specificities. Thus, two DQα RFLPs correlate with a single DQ serotype (DQα 1a and DQα 1c with DQw1), but three DQα RFLPs correlate with more than one DQ serotype (DQα1b with DQw1 and DQw3; DQα2 with DQw2 and DQw3; DQα3 with DQw2 and DQw3). Individual DQβ and DQα RFLP subtypes appear to correlate with single, or associated HLA-DR specificities. Specific combinations of DQβ with DQα RFLPs also correlate with HLA-Dw splits of DR2 and DRw6. A system for HLA-DNA typing is described, which uses RFLP patterns generated by sequential hybridization of TaqI-digested DNAs with short DRβ, DQβ and DQα cDNA probes. The DQβ and DQα probes not only identify the DQ allele, but because of linkage disequilibrium with DR, help to assign the DR allele, which may not always be identified with a DRβ probe alone.

HLA DQα and DQβ restriction fragment length polymorphisms associated with Felty's syndrome and DR4-positive rheumatoid arthritis

Abstract HLA DQα and DQβ cDNA probes were used to study TaqI generated restriction fragment length polymorphisms (RFLPs) in DR4-positive patients with Fetlys syndrome (FS), seropositive rheumatoid arthritis (RA), and in HLA-DR4 positive controls. The results of this analysis revealed two DQβ RFLP patterns (DQβ3a and DQβ3b) associated with DR4, of which DQβ3b was found at significantly higher frequency in patients with FS (73%) or with RA (52%) than in DR4 controls (29%). Hind III generated RFLPs provide evidence that DQβ3b is in strong linkage disequilibrium with the gene encoding the serologically recognized epitope TA10. Results obtained using a DQα chain probe revealed polymorphic difference between DQα chain genes associated with different DR types, thereby providing a possible explanation for the lack of association between RA and other DR haplotypes in linkage disequilibrium with TA10. We conclude that both DQα and DQβ genes may be important in determining HLA-linked susceptibility to serve forms of RA.

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.

Identification of Dw2, Dw12, and “Short” DR2 Splits with Sequential Exon-Specific DRβ, DQβ, and DQα cDNA Probes

The HLA-DR2 specificity has been divided by alloantisera (DR2 and “short” DR2) and by T cells (Dw2, Dw12, DB9 (LD-5a), FJO, and LD-MN2). The FJO and LD-MN2 splits appear to correlate with the short DR2 serotypes. Here, we report the correlation between the phenotypically recognized splits of DR2, and genotypes as defined by restriction fragment length polymorphism (RFLP) analysis. Genomic DNAs from 84 DR2-positive individuals and 10 homozygous typing cells were hybridized sequentially with short (exon-specific) cDNA probes isolated from the following clones: pRTV1 (DRβ), 512bp PstI fragment; pII-β-1 (DQβ), 627bp AvaI fragment; pDCH1 (DQα), 797bp PstI fragment. Methods and interpretation of DR and DQ alleles were detailed previously (1). Figure 1 shows the DRβ, DQβ, and DQα RFLP patterns observed in DR2-DQw1 haplotypes, either shown to segregate in family studies, or predicted from association due to linkage disequilibrium. Each split of DR2 defined by phenotypic criteria is associated with a particular combination of RFLPs for DRβ (DRβ2 or DRβ2sh), DQβ (DQβ1a, DQβ1b or DQβx), and DQα (DQα1b or DQαlc). Thus, of 121 DR2-DQw1 RFLP combinations examined, 105 were DRβ2-DQβ1b-DQαlb. This combination has been previously reported in DR2-Dw2 splits; four were DRβ2-DQβx-DQα1c, previously reported in DR2-Dw12 splits; nine were DRβ2sh-DQβ1a-DQαlb, which has been described in the ‘short’ DR2 splits FJO and LD-MN2, and was shown in this study to segregate within one family. FJO and LD-MN2 are reported to possess indistinguishable RFLPs (2) and identical nucleotide sequences for the β1 exon of the DRβ1 gene (3). The remaining novel RFLP combination, DRβ2-DQβxDQαlb, designated “DR2-Akhtar,” was uncovered in three Asian individuals from a single family and may be related to DB9 (LD-5a) based on similarities with published DQβ RFLP data, but shows the “long” DR2 DRβ RFLP (DRβ2), whereas the DB9 (LD-5a) DRβ RFLP as reported does not correspond to either DRβ2 or DRβ2sh (2,4). Investigation of DR2 haplotypes from other non-Caucasoid populations is likely to reveal further novel combinations of DRβ, DQβ, and DQα RFLPs.

Restriction Fragment Length Polymorphism of HLA-DR7 Alleles and Association with HLA-B Antigens

Although HLA-DR7 appears to be serologically homo- 2. geneous (1), it may be split according to epitopes recognized by T cells (Dw7, Dwl1) and is normally found in association with either DQw2 or DQw3. This report describes the correlation between restriction fragment 3. length polymorphism (RFLP) and phenotypic splits of DR7. Genomic DNAs from 74 DR7-positive individuals and from eight DR7-homozygous typing cells were analyzed by sequential hybridization with short (exonspecific) DRβ, DQβ, and DQα cDNA probes (J. L. Bidwell et al, this volume). Figure 1 shows the RFLP patterns revealed by each of these probes.

Antigen Society #8 Report (Bw70, Bw71, Bw72)

The Bw6-associated antigens Bw71 (Bu) and Bw72 (SV) were originally identified at low frequencies in English and Dutch Caucasoid populations (1,2). That Bu and SV were two closely related but discrete cross-reacting antigens was established in a two-center collaborative study (3) and in an investigation of East African blacks (4). The associations of A28 and Cw3 with Bu and of A23 and Cw2 with SV were confirmed in a further study of an East African black population (5).

A rapid solid-phase rosette-inhibition assay for the detection of Fc receptor (FcRIII)-blocking alloantibodies

Abstract A procedure for the detection of FcR-blocking alloantibodies is described which uses human B lymphocytes immobilised on plastic by poly- l -lysine. Antibodies which inhibited rosette formation between B lymphocytes or Daudi cells and ox erythrocytes coated with rabbit antibodies (EA) were detected in 10 out of 10 sera containing anti-HLA A2 antibodies and 3 out of 3 sera containing anti-HLA class II antibodies. Inhibition of rosette formation (EAI activity) was mediated by protein G-separated IgG. Analysis of rosette formation using these 13 sera and lymphocytes from 39 donors revealed that the degree of inhibition was bimodal; most sera were either clearly inhibitory or non-inhibitory in the assay. However, there was no correlation between inhibition of rosette formation (EAI activity) and lymphocytotoxicity. Four pairs of sera showed similar patterns of reactivity ( r > 0.6, p r = −0.86, p