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

HLA-DR and -DQ serotyped cell lines and peripheral blood leucocytes were analysed by Southern blot allogenotyping. Using a short DQ beta cDNA probe, a DQ beta allelic series was defined by restriction fragment length polymorphism (RFLP) with the restriction endonuclease TaqI. This DQ beta allelic series correlates with, and defines splits of, the HLA-DQ serological specificities DQw1 (DQ beta 1a and DQ beta 1b RFLPs), DQw2 (DQ beta 2a and DQ beta 2b RFLPs) and DQw3 (DQ beta 3a and DQ beta 3b RFLPs). By sequential use of a short DQ alpha cDNA probe a second, DQ alpha allelic series is defined by RFLP. This series correlates to a lesser extent than DQ beta RFLPs with the HLA-DQ serological specificities. Thus, two DQ alpha RFLPs correlate with a single DQ serotype (DQ alpha 1a and DQ alpha 1c with DQw1), but three DQ alpha RFLPs correlate with more than one DQ serotype (DQ alpha 1b with DQw1 and DQw3; DQ alpha 2 with DQw2 and DQw3; DQ alpha 3 with DQw2 and DQw3). Individual DQ beta and DQ alpha RFLP subtypes appear to correlate with single, or associated HLA-DR specificities. Specific combinations of DQ beta with DQ alpha 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 DR beta, DQ beta and DQ alpha cDNA probes. The DQ beta and DQ alpha 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 DR beta probe alone.     

Southern hybridization analysis of DNA polymorphism in the HLA-D region

Restriction fragment-length polymorphisms (RFLP) were systematically analyzed by Southern hybridization with restriction endonuclease-digested genomic DNA from 28 HLA-homozygous B cell lines with Dw1-Dw19 specificity using the DR beta and DQ beta chain cDNAs as probes. These probes detected polymorphic fragments unique to each HLA-DR specificity. Furthermore, the DQ beta chain probes permitted us to distinguish between different Dw specificities with an identical DR type much more efficiently than with the DR beta chain probe. Distribution analysis of restriction fragments hybridizing to DR beta in relation to the DR and DQ specificities showed several sets of them forming ten clusters, some of which correlate with DRw53, DQw1, and DR alleles. This DNA typing technique allows the direct definition of HLA types at the gene level and provides a powerful tool for isolating genes controlling HLA-associated diseases.     

Analysis of MHC class II DNA polymorphisms in Negroid subjects

Study of the MHC class II region is complicated by strong linkage disequilibrium between DR and DQ. Comparison of DR-DQ haplotypes between different races partly resolves this problem. We present the results of an analysis of DRB, DQA and DQB restriction fragment length polymorphisms in serologically DR-typed subjects of Negroid origin. Clearly distinguishable DRB RFLPs were observed for DR1,2,5,7 and w8. DR4,9 and w10 were uncommon in this group. DR3 was associated with two extended haplotypes, one characterised by the DQw4 allele, the other by the DQw2 allele. A recently recognised DQB RFLP (DQB 2c) was associated with DR7 and also occurred on DR5 and DR9 haplotypes. Both DR5 and DRw6 were heterogeneous in their DR-DQ relationships. Negroid subjects exhibit DR-DQ relationships distinct from other races. These provide scope for further study of MHC class II associations with disease.     

The complexity of DRw6 and DR5 haplotypes in American blacks demonstrated by serology, cellular typing, and restriction fragment length polymorphism analysis

This study describes the diversity of DRw6 and DR5 haplotypes in the American black population using serology, cellular typing, and restriction fragment length polymorphism (RFLP) analysis. DRw6 (DRw13 and DRw14) and DR5 (DRw11 and DRw12) haplotypes are observed at a high frequency in this population (DRw6: 32%, DR5: 30%). Many of these haplotypes express undefined HLA-D specificities and unusual DQ and DRw52 associations which previously have not been well characterized or reported (e.g., DRw13, DQw5, DRw52c, D-; DRw13, DQw2, DRw52a, D-; DRw11, DQw5, DRw52c, D-). Serologic analysis of class II alleles in American blacks suggests the presence of DRw13, DRw11 and DQw6 allelic variants and demonstrates the difficulty in defining DRw6 and DR5 in this population. The class II genes from four American black families expressing many of the novel DRw13, DRw14, DRw11, and DRw12 haplotypes defined by serology and mixed leukocyte culture were further characterized by RFLP analysis. The data presented here along with other published data identify at least eight DRw13 haplotypes (DRw13A-DRw13H) in the human population. Five of these haplotypes exhibit an undefined HLA-D specificity. Three DRw14 haplotypes (DRw14A-DRw14C) and eight DR5 haplotypes (DRw11A-DRw11E and DRw12A-DRw12C) were also identified. The novel DRw6 and DR5 haplotypes observed in American blacks may arise from differences in DRB1, DQA1, and DQB1 genes as well as from differences in the combinations of alleles of these genes encoded by a haplotype. The serologic and RFLP analyses suggest that some DRw13 and DRw11 haplotypes represent transitional steps between DRw13 and DRw11 in the evolutionary pathway which generated the DRw52 family.     

Extended HLA-DQw2 haplotypes: molecular analysis.

The HLA-DQw2 specificity, homogeneous in serology, is strongly associated to two HLA-DR specificities: DR3 and DR7. These alleles are found mainly on DQw2 bearing extended haplotypes with strong linkage disequilibrium. We describe, with BamHI, HindIII and RsaI, two restriction fragments length polymorphisms (RFLP) for the A gene of DQw2. These two subtypes correlated with the DR3 and DR7 specificities. Interestingly, by non-equilibrium pH gradient electrophoresis (NEPHGE), two DQ alpha chains were also found, respectively correlated with the same DR specificities. In addition, HincII polymorphism allowed us to distinguish several patterns of B genes for (DR7) DQw2 haplotypes but without any detectable association with another HLA marker. However, only one DQ beta chain was found by NEPHGE in the (DR7) DQw2 haplotype. Furthermore, HincII discriminated the B genes of the two extended haplotypes: (B8, DR3) DQw2 and (B18, DR3) DQw2. The same result was found by NEPHGE: two DQ beta chains were described, corresponding to the same extended haplotypes. The use of exon-specific DQB probes showed that the genomic polymorphism in DQw2 haplotypes is located, at least, at the 3' end of the gene. These data add new characteristics to the different DQw2 extended haplotypes.     

HLA-DR and DQ DNA polymorphisms in subjects of Asian Indian and white Caucasian origin

There is a close correspondence between serologically defined DR types and DR beta chain restriction fragment length polymorphisms (RFLPs). There is also an association between DR types and DQ alpha and DQ beta RFLPs because of linkage disequilibrium. We present the results of an analysis of DR beta, DQ alpha and DQ beta RFLPs in Asian Indians and white Caucasian subjects. DR beta RFLPs were similar in the two groups. Clearly distinguishable DR beta patterns were observed for DR1, 2, 3, 4, 5, 7 and w10. The DR beta patterns associated with DR3 were, however, also found with w6. The DR7 DR beta patterns were also found with w9. For DR specificities 1, 3, 4, 5, 7 and w10, the associated DQ alpha and DQ beta RFLPs were similar in both racial groups, but for DR2, however, marked differences were found. The DR2-positive white Caucasian subjects all possessed a single DQ alpha/DQ beta combination whereas the DQ alpha/DQ beta patterns in DR2-positive Asian Indians showed considerable heterogeneity. The pattern seen in white subjects was present in only a minority of Asians. DR-DQ relationships clearly vary in different racial groups. RFLP analysis of HLA-linked diseases in different populations should prove to be an important technique in identifying the primary genetic factor(s) in these disorders.     

Individuals with HLA-DR blank alleles display well-known DR-DQ RFLPs

We have characterized HLA-DRB, -DQA, and -DQB gene polymorphism in a large number of serologically DR blank haplotypes with the restriction enzymes Taq I, Bam HI, and Pvu II, with the aim of finding new RFLPs in the Caucasian population. Locus-specific RFLPs were combined for a definition of Taq I DR-DQ haplotypes. All observed DNA haplotypes could be found in a control group of 100 individuals, but with a different distribution. Serologically less well-defined specificities were over-represented in the blank group, in particular the DRw13-associated Taq I DR-DQ haplotype T-13.3. We conclude that the majority of DR blank haplotypes are probably closely related or identical to previously defined DR alleles. The extent of DR polymorphism in the Caucasian population seems to be well mapped, considering the extremely small proportion of rare Taq I DR-DQ haplotypes and lack of new patterns in this study.     

Analysis of DR and DQ gene products of the DR4 haplotype in patients with IDDM: Possible involvement of more than one locus

Fifteen DR4-bearing haplotypes from twelve patients with insulin-dependent diabetes mellitus (IDDM) were analyzed serologically, cellularly, and biochemically. The HLA-Dw composition of these DR4-positive haplotypes was Dw4 (46%), Dw14 (22%), and Dw10 (33%). The biochemical analysis by two-dimensional electrophoresis (2D-PAGE) of the DR beta chains showed that each Dw specificity is characterized by a specific DR4 beta chain that appears to be identical in normal and diabetic individuals. Analysis of DQ beta chains in the DR4-bearing haplotypes revealed that certain Dw specificities such as Dw4 are characterized by the presence of either the DQw7 (formerly DQw3.1) or DQw8 (formerly DQw3.2) alleles, which generate the Dw4.1 or Dw4.2 subtypes, respectively. Others such as Dw14 and Dw10 are characterized by the presence of the DQw8 allele. In our sample of 12 patients the Dw4.2 (Dw4, DR4βI-4 DQw8) and Dw10 (Dw10, DR4βI-1, DQw8) subtypes were predominant. It is concluded that individual DR β and DQ β gene products from the DR4-bearing haplotype of IDDM patients are identical to those of normal control subjects and that Dw14 as well as Dw10 are involved in disease susceptibility. We suggest that disease susceptibility may be influenced by more than one locus within the HLA-D region.     

HLA-DR and DQ DNA genotyping in multiple sclerosis patients in Northern Ireland.

DNA from multiple sclerosis (MS) patients and healthy control individuals from the Northern Ireland population, was assessed by restriction fragment length polymorphism (RFLP) analysis to look for disease-associated polymorphisms. HLA-DR and -DQ allogenotyping was performed using a single enzyme (TaqI)/Multiple probe system. The TaqI/DR beta RFLPs correlate well with serologically defined specificities and in addition detect further subtypes of these associated with DQ or Dw specificities. The results confirm an association of MS with DR beta 15 and show a decreased frequency of DR beta 4 in the patients. An increased frequency of DR beta 17 in patients negative for DR beta 15 was also found. There is a decrease in the frequency of the TaqI/DQ beta 3 (TA10 + ve) RFLP and the TaqI/DQ alpha 2LL genotype in the MS patients, these not being associated with DR beta 15. In addition RFLP analysis of the DPA1 and DPB1 genes reveals an MspI/DP beta 3.0-kb fragment which occurs at a higher frequency in the patients than in the controls.     

Studies of RFLP-inferred HLA-DR-DQ haplotypes in Danish women with recurrent fetal losses

Results of HLA-DR and -DQ typing by RFLP in 152 unrelated women with at least 3 unexplained fetal losses were compared with those of 210 normal controls. The overall distribution of DR-DQ phenotypes did not differ significantly between patients and controls. In a subgroup of 59 patients having had at least 4 fetal losses, a significantly higher number of patients than controls were DRw17,DQw2-positive (RR = 2.9, pcorrected less than 0.02). DR-DQ haplotypes which carry DQB1 alleles encoding an amino acid other than aspartic acid at codon 57 in the second exon (non-Asp57 haplotypes) have been reported to confer susceptibility to several immunologically-mediated diseases. We found that the total frequency of non-Asp57 haplotypes (other than DR7,DQw2 which has unique features) was significantly increased in patients (50.0%) compared with controls (39.3%) (p less than 0.005). In the total group of patients and in the group with at least 4 fetal losses, 22.4% and 32.2% respectively were homozygous non-Asp57/non-Asp57 compared with 13.3% among controls (RR = 1.9, p less than 0.025 and RR = 3.1, p less than 0.001, respectively). The results suggest that DRw17,DQw2 confers susceptibility to suffer recurrent fetal losses. However, it is possible that the HLA-associated susceptibility may be primarily conferred by DQ molecules lacking aspartic acid at codon 57 in the DQ beta chain.     

Cells homozygous for two HLA-DR7-associated HLA-D specificities express highly similar DR molecules but different DQ molecules

The Ia molecules expressed by cells homozygous for two distinct HLA-DR7-associated HLA-D specificities, Dw7S and Dw11L, were compared. The complete Ia phenotypes of these cells are DR7, DRw53, DQw2, Dw7S, DPw4 and DR7, DRw53, DQw3, Dw11L, DPw4, respectively. Immunofluorescence analysis revealed that three DQ-specific monoclonal antibodies (Leu-10, 33.1, and HK-19), which detect polymorphic DQ determinants that do not correspond to known serologic specificities, are nonreactive with DR7, Dw7S cells but are reactive with DR7, Dw11L cells. The DR molecules isolated from Dw7S and Dw11L cells are very similar and comigrate when analyzed together by two-dimensional gel electrophoresis. In contrast, the DQ molecules isolated from these cells are structurally distinct: the DQ beta chains of DQw2-bearing molecules from Dw7S cells are very basic, while those of DQw3-bearing DQ molecules from Dw11L cells are more acidic. The finding that two DR7, D-different cells express indistinguishable DR molecules and structurally distinct DQ molecules documents a unique pattern of Ia molecular organization which is different from those previously described for the DR2-, DR4-, or DRw8-associated HLA-D specificities.     

Definition of DRw10 specificity by restriction fragment length polymorphism

The purpose of this study was the RFLP characterization of the DRw10 specificity. Twenty-two DRw10 cells were tested: the DNAs were digested by seven restriction enzymes and hybridized with DR beta, DQ beta and DQ alpha probes. Hybridization with DR beta revealed a pattern particular to the DRw10 specificity, with a specific TaqI 12.5Kb fragment. Hybridization with both DQ-specific probes showed that DRw10 is always associated with a special DQw1 subtype: DQw5. Furthermore, at DR and DQ levels, the 22 DRw10 cells behaved homogeneously.     

Increasing complexity of HLA-DR2 as detected by serology and oligonucleotide typing.

Serological and oligonucleotide typing was performed on a number of HLA-DR2-positive cells from different ethnic origin, including DR2 haplotypes with various DQ associations. Exons 2 of DRB1 and DRB5 of DR2-positive individuals were locus-specific amplified and hybridized with a number of different oligonucleotides capable of discriminating between the various Dw2, Dw12, Dw21, and Dw22 associated sequences. The linkage of DRB with DQA1 and DQB1 in these haplotypes was analyzed. Among the DR2- positive cells we could define 10 different DR DQ haplotypes by serology and 13 by oligonucleotide typing. The DR2.ES specificity is a serological DRw15 variant which could not be discriminated by oligonucleotide typing from a DRw15 DQw5 haplotype. The DR2.JA variant represents a unique DRB1*1602 DRB5*0101 haplotype. The DR1+2s haplotype consists of a DRB1 DQ region from a Dw1 and a DRB5 gene from a Dw2 haplotype. Its short DR2 serum pattern can be explained by the absence of a DR2 DRB1 gene product. DRB5*0101 sequences were found in association with DRB1*1501, *1502, *1602, and *0101 alleles. Since the DRB5 gene is capable of such different associations it is comparable to the DRB3 and DRB4 genes. This may have implications for the definition of the broad DR2 specificity which is predominantly encoded by the DRB5 gene product. New DR2 haplotypes included the following DQ combinations: DQw2-positive DQA1/B1*0301/0201 and DQw6-positive DQA1/B1*0102/0601 and *0102/0603 haplotypes.     

Unusual HLA-DR,DQ haplotypes found in South African families of black, Asian Indian, and mixed ancestral origin.

Four non-Caucasoid families with the unusual HLA-DR,DQ haplotypes DRw17,DQw7; DR9,DQw2; DR4,DQw2; and DR4,DQw5 were studied. All four haplotypes showed identical serological patterns to those seen with the equivalent Caucasoid antigens, but no HLA-Dw specificities could be assigned. TaqI restriction fragment length polymorphism (RFLP) patterns observed using DRB, DQB, and DQA probes showed that the DRw17,DQw7 haplotype may have originated from a homologous crossover between a DRw17,DQw2 haplotype and a haplotype with DQw7. The results obtained for the DR9,DQw2 and DR4,DQw2 haplotypes suggest that these could have resulted from recombination events with an ancestral "black" DQw2 haplotype. From the RFLP data, it is difficult to postulate the origin of the DR4,DQw5 haplotype being from a single recombination event.     

Biochemical dissection of DRw6 related epitopes using monoclonal antibodies

Three monoclonal antibodies (MoAbs) directed against polymorphic epitopes of Ia antigens were used as tools for a serological and biochemical dissection of the class II products encoded by the HLA-DRw6 haplotype. MoAb 16.23 defines an epitope common to DRw13 and DR3 haplotypes, MoAb S5 defines an epitope common to DRw13 and DR2 haplotypes, and MoAb S2 defines an epitope apparently restricted to DRw13. Not all DRw13 cells express these epitopes. Analysis of 16 DRw6 homozygous typing cells showed that expression of all three epitopes was restricted to those DRw13 cells that carried the Dw18 antigen, the DRw13 Dw19 cells being negative. The relationships among the molecules bearing these epitopes were investigated using sequential immunoprecipitation in lymphoblastoid cell lines derived from genetically characterized individuals. In both DRw13 and DR2 bearing cells, the DR2 + w13 epitope was localized to a population of DQ molecules which also carried the DQw1 specificity defined by MoAb Genox 3.53. The S5 epitope is therefore a private specificity that distinguishes the DQw1 antigens encoded by the DR2 and DRw13 haplotypes from the DQw1 antigens encoded by other haplotypes. The DRw13 and the DR3 + w13 epitopes were both shown to be expressed on DR molecules that also carried a DRw52-like specificity. In a DRw13 haplotype encoding both the S2 and 16.23 epitopes, the epitopes appeared to be located on separate molecules. The antibodies described here can distinguish between DRw13 cells which carry different Dw antigens, identify a private specificity on the DQw1 antigen, and define two distinct DR molecules encoded by some DRw13 haplotypes.     

HLA-D typing of heterozygotes using restriction fragment length polymorphism in the HLA-DQ gene region on the basis of standard band patterns derived from HLA homozygotes

Genomic DNAs from 37 Epstein-Barr virus-transformed HLA-D homozygous cell lines with different Dw specificities, Dw1-Dw23, and DKT2 were digested with four different restriction endonucleases (EcoRI, PstI, TaqI, and MspI) and hybridized to DQ alpha and DQ beta cDNA probes. Polymorphic patterns of multiple fragments correlating with Dw, DQ, and DR haplotypes were detected, and the restriction fragment length polymorphism standard pattern specific for each Dw, DQ, and DR specificity could be defined. The polymorphic fragment patterns of HLA-D heterozygotes were predicted simply by the summation of two standard patterns of HLA-D homozygotes and utilized to identify HLA-D specificities of 25 normal individuals, who are HLA heterozygotes in most cases. The HLA-D, -DR, and -DQ specificities defined by this DNA typing were compared with those assigned by serologic and cellular typing. There was good correlation, allowing the application of accurate genotyping by DQ alpha and DQ beta cDNA probes to HLA class II typing of HLA heterozygotes.     

Extensive study of DRB, DQA, and DQB gene polymorphism in 23 DR2-positive, insulin-dependent diabetes mellitus patients.

To gain insight into the HLA subregions involved in protection against insulin-dependent diabetes mellitus (IDDM) we investigated the polymorphism of HLA-DR and -DQ genes in 23 DR2 IDDM patients. Results show the following. (1) Fourteen patients (61%) possess the DRB1, DRB5, and DQB1 alleles found in DRw16/DQw5 healthy people. These data contrast with the 5% of DRw16 normally found in DR2 populations and are in agreement with former observations supporting that the DRw16 haplotype is not protective. (2) Nine DR2 patients, i.e., 39% versus 95% in published DR2 controls, possess the DRB alleles found in DRw15 unaffected people. Among them, six patients have also DQA1 and DQB1 alleles identical to those found in DRw15/DQw6 healthy individuals. These data confirm that the DRw15/DQw6 haplotype is protective but indicate that none of the DR or DQ alleles, alone or in association, confers an absolute protection. (3) Our most striking results concern the very high frequency of recombinant haplotypes among the DRw15 patients: 3 of 9. In these three patients recombinations led to the elimination of both DQB1 and DQA1 alleles usually associated with DRw15. This strongly suggests that the occurrence of IDDM in these DRw15 patients is due to the absence of the usual DQ product and thus reinforces the assumption that DQ rather than DR region is involved in the protection conferred by the DRw15/DQw6 haplotype. Finally, analysis of the non-DRw15 haplotypes in heterozygous patients showed that IDDM can occur in the absence of any DQ alpha beta heterodimer of susceptibility.     

Multiple epitopes on a single DQ molecule from the DQw3-carrying haplotypes

Multiple polymorphisms on the DQ molecule(s) have been detected by monoclonal antibodies (MoAbs). Among these, TA10 and IIB3 have been described as two new alleles in the DQ region other than the conventional DQw1-w3 allelism. The TA10 specificity is DQw3-related and is in linkage disequilibrium with DR5 and weakly associated with DR4. The IIB3 specificity is DQw1-related and is in linkage disequilibrium with DR2, DR4, DRw9 and DRw13. Thus, the DQw3-carrying haplotypes are either positive with TA10 or IIB3. The molecular and topological analysis has revealed that both TA10 and IIB3 determinants were expressed on a single DQ molecule that also carried the DQw3 determinants on DR5 and DR4 cell lines, respectively. Thus, a single DQ molecule generated multiple epitopes detected by alloantisera and/or MoAbs at least on the DQw3-carrying haplotypes. These would be useful for unraveling the largely unknown functions of the DQ class II molecules.     

Identification of HLA-DRw52 associated antigens using HLA class II allogenotyping

The HLA-DR antigens DRw8, DRw11, DRw12, DRw13, DRw14 and DRw17 are strongly associated with the supertypic specificity DRw52. This association has been used to assist in assignment of serological specificity. However, difficulties in the identification of these antigens arise since they are serologically crossreactive. This report describes the application of restriction fragment length polymorphism (RFLP) allogenotyping to assist in the positive identification of DRw8, DRw11, DRw12, DRw13, DRw14, DRw17, DRw52a and DRw52b, and in addition describes further subtypes defined by RFLP which correlate with DQ or Dw associations. We also describe a novel TaqI/DR beta RFLP in a Caucasian family which types serologically as DRw12.     

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.