RFLP analysis of HLA-DR and -DQ genes and their linkage relationships in the Pacific

Human genomic DNA samples from Melanesians, Micronesians, and Caucasoids of known HLA-DR type were examined with cDNA probes for HLA-DR alpha, -DR beta, -DQ alpha, and -DQ beta chain genes. DR beta hybridizations with TaqI-digested DNA did not detect any new DR specificities in the Pacific. However, within the DR5 specificity a common DNA subtype was found in Pacific Islanders that was not seen in Caucasoids. Altogether, four DNA subtypes of DR5 are described. With the DQ alpha and DQ beta probes, significantly more variation could be demonstrated between populations. For example, DR2 was associated with a DQ beta TaqI pattern in the Pacific that was very rare in Caucasoids and additional RFLP analysis with other enzymes showed that this pattern is probably associated with the Dw12 subtype of DR2. DRw8-positive samples showed two different DQ alpha TaqI patterns, and these correlated with DQw1 and DQw3 specificities. DR alpha hybridizations with BglII-digested DNA also revealed different linkage relationships of the HLA-class II region genes between Pacific and Caucasoid specimens. The different population linkage disequilibrium relationships have permitted tentative assignment of TaqI fragments to either the DR beta 1 or DR beta 2 genes and are highly suggestive that the DQw1 specificity is encoded by the DQ alpha chain gene. This study shows the value of population comparisons in contributing to knowledge of the genetic organization of the genome.     

A novel association of DQ alpha and DQ beta genes in the DRw10 haplotype. Determination of a DQw1 specificity by the DQ beta-chain

The association of the class II genes of the DRw10 haplotype from a cell line, NASC, initiated from a member of a well characterized family, was analyzed by sequencing cDNA clones corresponding to DR beta I, DQ alpha, and DQ beta genes. An identical haplotype was also identified in the Raji cell line. In addition to typing as DRw10 and DQw1 with HLA typing sera both, the NASC and Raji cell lines were shown to react strongly with the monoclonal antibodies 109d6 (specific for DRw10 beta 1 and DRw53 beta 2 gene products) and Genox 3.5.3 (specific for DQw1) and exhibited the restriction fragment length polymorphism indicative of a DRw10, DQw1 haplotype. The DR beta 1 gene corresponding to the DRw10 specificity was found to have a first domain sequence different from all other DR beta I genes. Sequence analysis of the 3'-untranslated region of this DR beta-chain gene showed a significant divergence from the 3' untranslated region of the DRw53 family of haplotypes and a lesser divergence from that of the DRw52 and DR1/DR2 families. The sequence of the DQ beta genes corresponding to the DQw1 specificity in the DRw10 haplotype was found to be identical to the DQ beta gene from a DR1, DQw1 haplotype. Surprisingly, however, the DQ alpha gene did not resemble other DQw1-like DQ alpha genes, but was identical in sequence to the DQ alpha gene found in DR4 haplotypes. The novel association of DQ alpha and DQ beta genes in the DRw10 haplotype revealed in these studies may result from a double recombinational event. More consequentially, these studies strongly suggest that the DQw1 specificity recognized by Genox 3.5.3 is determined by the DQ beta chain and is not affected by the DQ alpha-chain.     

Analysis of the molecular specificities of anti-class II monoclonal antibodies by using L cell transfectants expressing HLA class II molecules

Expressible HLA class II alpha- and beta-chain cDNA were used for DNA-mediated gene transfer to produce L cell transfectants expressing single types of human class II molecules. Cloned transfectants expressing nine different class II molecules were isolated: DR alpha: DR1 beta I, DR alpha: DR4 beta I, DR alpha: DR5 beta I, DR alpha: DR5 beta III (DRw52), DR alpha: DR7 beta I, DR alpha: DR4/7 beta IV (DRw53), DQ7 alpha: DQw2 beta, DQ7 alpha: DQw3 beta, and DPw4 alpha: DPw4 beta. These class II-expressing transfectants were used to analyze by flow cytometry the molecular specificities of 20 anti-class II mAb. These analyes indicate that some mAb are more broadly reactive than was previously thought based on immunochemical studies. In contrast, the narrow molecular specificities of other anti-class II mAb were confirmed by this approach. Transfectants expressing human class II molecules should be valuable reagents for studies of B cell and T cell defined epitopes on these molecules.     

Recombination sites in the HLA class II region are haplotype dependent

We have analyzed DNA sequence polymorphisms of DQ alpha and DQ beta chains from three haplotypes from the DRw52 family: DR5 DQw1 (FPA, GM3106), DRw6 DQw1 (CB6B, 10w9060), and DRw6 DQw3 (AMALA, 10w9064). The results indicate that the DR5 DQw1 and DRw6 DQw1 haplotypes have arisen by recombination between the DR beta 1 and DQ alpha loci. This contrasts with our previous analysis of DR4 DQ"Wa", DR3 DQ"Wa", and DR7 DQw3 haplotypes, all of which appear to have arisen by virtue of recombination between DQ alpha and DQ beta. Thus, there appear to be at least two different sites where recombination has occurred within the DR and DQ subregions. These differing patterns of recombination were interpreted in the context of the three major family groups of class II haplotypes, the DRw53, DRw52, and DR1/2 haplotype families. The data indicate that haplotypes from these family groups tend to undergo recombination at different locations. We propose that these differences in site of recombination are a reflection of differences in the molecular organization of the haplotypes belonging to each family group.     

Polymorphism of the HLA-D region in American blacks. A DR3 haplotype generated by recombination

The polymorphism of HLA class II molecules in man is particularly evident when comparisons between population groups are made. This study describes a DR3 haplotype commonly present in the American black population. Unlike the Northern European population in which almost all DR3 individuals are DQw2, approximately 50% of DR3-positive American blacks express a serologically undefined DQ allelic product. DNA restriction fragment analysis with the use of several unrelated individuals and an informative family has allowed us to identify unique DQ alpha- and beta-fragments associated with the DR3, DQw- haplotype. Based on fragment size, the DQ alpha genes of the DR3, DQw- and DRw8, DQw- haplotypes are similar as are the DQ beta genes of DR3, DQw-; DRw8, DQw-; and DR4, DQw- haplotypes. In addition, a DX beta gene polymorphism has been identified which is associated with some DR3 haplotypes including the American black DR3, DQw- haplotype. cDNA sequence analysis has revealed a DQw2-like alpha gene and a DQ beta gene which is similar to that previously described for a DR4, DQw- haplotype. It is postulated that recombination between DQ alpha and DQ beta genes and between the DQ and DX subregions has generated the various DR3 haplotypes and has played an important role in creating diversity in the HLA-D region.     

Allelic forms of the alpha- and beta-chain genes encoding DQw1-positive heterodimers

On chromosome 6, in the HLA region, the DQ subregion is located immediately centromeric to the DR subregion. Even though only three serological specificities to date have been officially recognized (DQwl, DQw2, and DQw3), it seems likely that the phenotypical polymorphism expressed by DQ molecules is much more complex. There are reasons to believe that fixed alpha-beta combinations exist, each of them associated with a different DR allele. DQw1 is a determinant present on DQ molecules that are found associated with DRI-, DR2-, and DRw6-positive haplotypes. By restriction fragment length polymorphism analysis, we recognized three allelic DQ-alpha and three allelic DQ-beta patterns associated with DQw1 . In addition, one of these alpha/beta pairs associated with DR1, two with DR2, and a fourth with DRw6. We have obtained evidence using nucleotide sequencing that there are as many allelic forms of DQ-alpha and DQ-beta genes as there are different molecular DQ-alpha and DQ-beta patterns. The DQ-alpha and DQ-beta chains of DQwl-positive molecules each are encoded by at least three distinctly different allelic genes, and particular alpha/beta gene combinations are associated with the same DR alleles as their corresponding molecular alpha/beta pairs.     

Molecular analysis of the HLA class II genes in two DRw6-related haplotypes, DRw13 DQw1 and DRw14 DQw3

We have compared the sequence polymorphism of HLA class II genes of two distinct DRw6 haplotypes. cDNA libraries were constructed from two lymphoblastoid cell lines: CB6B (10w9060) which types as DRw13 DQw1, and AMALA (10w9064) which types as DRw14 DQw3. Multiple sequence differences were found at the DR beta I, DQ alpha, and DQ beta loci when these two haplotypes were compared. The DR beta I allele found in the DRw14 DQw3 haplotype appears to have diverged primarily as a result of a gene conversion event with a DR1 allele acting as donor. In contrast, the DRw13 DQw1 haplotype appears to have arisen by means of a recombination event between the DR and DQ subregions. Thus, multiple genetic mechanisms, including point mutation, gene conversion, and recombination, have generated diversity among DRw6 haplotypes.     

Complete sequence of the HLA DQ alpha and DQ beta cDNA from a DR5/DQw3 cell line

The HLA-D region is composed of three subregions termed DR, DQ, and DP. We previously reported the sequence of a DR5 beta I and two DR5 beta III cDNA from the DR5 cell line Swei. We now report on the nucleotide and deduced amino acid sequence of the DQ alpha and DQ beta cDNA from the same DR5 cell line, which also types as DQw3. Comparison with other available DQ sequences indicates that DQ alpha has one region of major variability, whereas DQ beta appears to have four regions of variability. In addition, these comparisons indicate that DQw3 alpha from DR5 is different from DQw3 alpha from DR4, but identical to DQw2 alpha from DR3. In contrast, DQw3 beta from DR5 is very similar to DQw3 beta from DR4. These data indicate that at least for DQw2 and DQw3 it is the DQ beta chain that is responsible for DQ typing. Most sequence differences in DQ alleles can be attributed to point mutations; however, codon additions/deletions in the DQ alpha chain may contribute to variability. In addition, regions of possible gene conversion in the DQ alpha and DQ beta chains is suggested by the presence of a chi-like sequence in each chain. Finally, comparison of available haplotypes suggest recombination events may take place between DQ beta and DQ alpha, between DQ alpha and DR beta I, and between DR beta I and DR beta III.     

Molecular mapping class II polymorphisms in the human major histocompatibility complex. II. DQ beta

The restriction fragment length polymorphisms have been determined for six restriction enzymes (Bam HI, Bg1 II, Eco RI, Hinc II, Hind III, and Pvu II) and a DQ beta probe on 25 cell lines that are homozygous by consanguinuity at the MHC. These patterns reflect both DR haplotypes and DQ types of the cells tested. At least one non-polymorphic band is present in all the cell lines with every restriction enzyme except Hinc II. This band most probably represents DX beta hybridization. The polymorphic bands indicate that more polymorphism exists in the DQ subregion than is predicted serologically. Each DR haplotype is associated with a unique set of restriction fragments except for DR2 and DR6. The patterns are largely consistent within each DR haplotype. In addition, some bands reflect the established DQ specificities DQw1 and DQw2. Individual bands can be identified that are unique to the haplotypes DR1, DR4, DR5, and DR6 and the DQw1- and DQw2-associated haplotypes. Subdivisions of haplotypes can be identified with this probe. In particular, MVL (DR1), Akiba (DR2), QBL (DR3), FPF (DR5), and APD (DR6) have polymorphisms that distinguish them from other members of their DR haplotype.     

HLA-DR typing "at the DNA level": RFLPs and subtypes detected with a DR beta cDNA probe

The HLA-DR beta gene, used as a hybridization probe, detects RFLPs that correlate with HLA-DR specificities. Using genomic DNA from more than 200 individuals, we have carried out a population study with a cDNA probe for the DR beta chain, which, under appropriate conditions, does not cross-hybridize with genes from other HLA-D subregions (e.g., DP and DQ). We first assessed the correspondence between serologically defined HLA-DR types and DNA patterns obtained after digestion with TaqI and found that DNA patterns allowed us to identify most specificities. Only two pairs of antigens are not distinguishable: with the DR beta probe alone we cannot distinguish DR3 from DRw6 or DR7 from DRw9. However, the correct assignment can always be made for the first pair by hybridizing the same digests with a DQ alpha or DQ beta probe. Thus DR typing from the DNA patterns is practical and accurate. We also looked for serologically undetectable subtypes. RFLPs revealed high-frequency subtypes for the specificities DR 2, 3, 5, w6, 7, and w9. Some of these are more accurately viewed as variant haplotypes, since the relevant variation is probably not at the DR beta locus that determines the serological specificities but rather at other closely linked and highly homologous DR beta loci such as DR beta-III. Nevertheless, the existence of variant haplotypes for so many specificities indicates a wealth of polymorphic variation beyond that detected serologically and provides more specific markers for studies of various diseases associated with HLA-DR specificities.     

Molecular variation of human major histocompatibility complex DQw3 beta-chains

Histocompatibility leukocyte antigen DQ molecules exhibit polymorphism of both DQ alpha- and beta-chains. Histocompatibility leukocyte antigen-DQw3 is associated with both DR4 and DR5 and can be further subdivided by reactivity with the monoclonal antibody TA10. To determine the molecular nature of the DQ polymorphic alleles associated with the DR4 haplotype, we have sequenced and analyzed DQ alpha and beta cDNA clones obtained from a DR4, Dw4, DQw3 cell line which is TA10-positive. The DQ alpha-chain sequence was identical to previously published sequences from the DR4 haplotype, but the DQ beta sequence differed from published DR4-DQ beta sequences obtained from DQw3-positive TA10-negative cell lines by eight amino acids, six of which were located in the beta 1 domain. Thus, the TA10 serologic determinants reside on the DQ beta-chain. A TA10-specific oligonucleotide probe was constructed based on the DQ beta sequence, and its specificity was confirmed in a panel of TA10-positive and TA10-negative cell lines. An additional band was observed in Southern blotting experiments which may indicate a donor sequence for gene conversion.     

Two divergent routes of evolution gave rise to the DRw13 haplotypes

The HLA class II genes and haplotypes have evolved over a long period of evolutionary time by mechanisms such as gene conversion, reciprocal recombination and point mutation. The extent of the diversity generated is most clearly evident in an analysis of the HLA class II alleles present within DRw13 haplotypes. This study uses cDNA sequencing to examine the first domains of DRB1, DRB3, DQA1, and DQB1 alleles from several American black individuals expressing seven different DRw13 haplotypes, five with undefined HLA-D specificities (i.e., not Dw18 or Dw19). Two new DRw13 alleles described in this study are the first examples of convergent evolution of DR alleles in which gene conversion has apparently combined segments of DRB1 alleles encoding DRw11 and DRw8 to generate two new DRB1 alleles, DRB1*1303 and DRB1*1304, that encode molecules bearing serologic determinants of a third allele, DRw13. These new DRw13 alleles are found embedded in haplotypes of DRw11 origin distinct from haplotypes encoding previously identified DRw13 alleles, DRB1*1301 and DRB1*1302. These data suggest that two evolutionary pathways may have given rise to two subgroups of alleles encoding molecules that share DRw13 serologic determinants yet which possess different structural and, likely, functional motifs. Reciprocal gene recombination events resulting in different DR, DRw52 and DQ allele combinations also appear to have played a crucial role in augmenting the level of diversity found in DRw13 haplotypes. Recombination has resulted in the association of one of the new DRw13 alleles with a DQw2 allele normally found associated with DR7 and the association of the DRw52c-associated DRw13 allele (DRB1*1302) with three different DQw1 alleles. The seven DRw13 haplotypes that have resulted from the effect of recombination on haplotypes formed by the two pathways of DRw13 allelic diversification have resulted in different repertoires of class II molecules and, most likely, different immune response profiles in individuals with these haplotypes.     

Analysis of DR beta and DQ beta chain cDNA clones from a DR7 haplotype

A cDNA library was constructed from a DR7, DRw53, DQw2 homozygous cell line, cDNA clones corresponding to DR beta and DQ beta chains were isolated, and the nucleotide sequences of the polymorphic first domains of these chains were determined. A novel screening strategy allowed rapid and simple identification of cDNA clones corresponding to both DR beta chains (DR7 beta1 and DR7 beta2): DR7 beta2 clones have a recognition site for the enzyme BssHII, whereas DR7 beta1 clones do not. The DR7 beta 1 sequence differs significantly from all previously described DR beta chains. As predicted by the presence of the BssHII site in DR7 beta 2 clones, the DR7 beta 2 sequence differs from the DR7 beta 1 sequence. The sequence of the DRw53-associated DR7 beta 2 chain is identical to the reported sequence of the DRw53-associated DR4 beta 2 chain. In addition, the sequence of the DQ beta chain from the DR7, DQw2 cell line is identical to the reported sequence of a DQ beta chain from a DR3, DQw2 cell. These findings raise interesting questions about the evolution of the DR3, DR4, and DR7 haplotypes.     

T lymphocyte recognition of a celiac disease-associated cis- or trans-encoded HLA-DQ alpha/beta-heterodimer

The HLA-associated susceptibility to develop celiac disease may to a large extent be attributed to the combination of particular DQA1 and DQB1 genes, i.e., the DQA1*0501 and DBQB1*0201 alleles, located either in cis position (on the DR3DQw2 haplotype) or in trans position (DR5DQw7/DR7DQw2 heterozygous individuals). We report three alloreactive T lymphocyte clones that recognize an HLA-DQ alpha/beta heterodimer both when the DQA1*0501 and DQB1*0201 alleles are located in cis or in trans position. Thus, the celiac disease associated DQA1 and DQB1 genes encode a functionally expressed DQ alpha/beta heterodimer.     

Molecular studies of a rare DR2/LD-5a/DQw3 HLA class II haplotype. Multiple genetic mechanisms in the generation of polymorphic HLA class II genes

A cDNA library was constructed from a homozygous B lymphoblastoid cell line (REM) obtained from an individual of a long isolated American Indian tribe, the Warao. The REM cell line expresses serologically defined determinants, DR2 and DQw3, and the T lymphocyte-defined (Dw/LD) specificity, LD-5a. T cells can recognize differences between FJO (a DR2/DQw1 cell line that expresses the Dw specificity MN2) and REM for both DR and DQ molecules. cNDA clones encoding the polymorphic DR beta 1-, DR beta 2-, DQ beta-, and DQ alpha-chains were sequenced and compared with other DR and DQ gene sequences. The DR beta 1-sequence of REM is identical to the DR beta 1-sequence of FJO; the DR beta 2-sequence is also identical to that of FJO except for one amino acid difference at position 67 in the polymorphic first domain (Leu in REM, Phe in FJO) due to a single point mutation. The DQ beta-sequence is identical to that of DR4/DQw3 haplotype; the DQ alpha is different from the DQ alpha of DR4/DQw3 haplotype and identical to the DQ alpha of both the DR3/DQw2 haplotype of a Raji cell line and the DR5/DQw3 haplotype in deduced amino acid sequence. Taken together, these findings suggest that: 1) a single amino acid difference (position 67) in the third hypervariable region of the first domain of the DR beta 2-chain in the DR2 haplotype is apparently sufficient for stimulating T cell responses; 2) the DQw3 serologic specificity may be defined mainly by the DQ beta-rather than DQ alpha-chain; and 3) multiple genetic events have probably occurred to generate the rarely found REM (DR2/LD-5a/DQw3) haplotype.     

Molecular analysis distinguishes two HLA-DR3-bearing major histocompatibility complex extended haplotypes

We detected restriction fragment length polymorphisms that distinguish the extended haplotype HLA-B8,DR3,SCO1 from HLA-B18,DR3,F1C30 at the DR beta and DQ beta loci with five of seven restriction endonucleases used. One set of restriction fragments was always found on HLA-B8,DR3,SCO1 and associated with DRw52a, while the other was present on HLA-B18, DR3,F1C30 and correlated with DRw52b (the gene encoding the subtype of DRw52 associated with the BO1 or LB-Q1 antigen). Furthermore, using a full-length DQ beta gene probe, we found division in the DQw2 haplotype, in which DQw2a always associated with HLA-B8, DR3,SCO1, while DQw2b always occurred with HLA-B18,DR3,F1C3O. Our evidence thus indicates that serologically defined HLA-DR3, HLA-DRw52, and HLA-DQw2 are each produced by two structurally very different sets of genes, one set occurring in HLA-B8, DR3,SCO1, and the other in HLA-B18,DR3,F1C30.Abbreviations used in this paper BSA bovine serum albumin - MHC major histocompatibility complex - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecyl sulfate     

Study of the HLA-DQ system by the complement fixation test on lymphocytes stimulated by phytohemagglutinin. Existence of HLA-DQX allele(s)

The complement fixation microtechnique against PHA blasts has been used to study HLA-DQw1, 2, 3 specificities with sera from multiple transfused patients and/or from multiparous women. Several sera (6 or 7) have been used to define each DQ specificity. The sera have been chosen because of their reactivity with cells from HLA-DR 1, 2 or w6 donors (for DQw1), DR3 or 7 donors (for DQw2,) DR4 or 5 donors (for DQw3). Correlation coefficients between DQ and DR specificities were from 0.56 to 0.91. Correlation coefficients between sera were from 0.51 to 0.92 in each cluster of sera. The segregation of DQw1, 2, 3 specificities has been studied in 46 families with 234 children. This study showed haplotypes lacking DQw1, 2, 3 specificities. The segregation of such 11 DQX haplotypes has been observed in 38 children from 8 families; 5 children were DQX/DQX homozygotes. Up to now, no serological reagent defining the specificity (or specificities) corresponding to DQX has been found. No preferential association was observed between DQX and DR specificities. The gene frequencies observed in 170 haplotypes in these 46 families were as follows: DQw1: 0.400; DQw2: 0.252; DQw3: 0.282; DQX: 0.065. Detecting DQ specificities seems easier by CF on PHA blasts than by lymphocytotoxicity microtechnique against B lymphocytes and monocytes from pheripheral blood. This suggests that PHA blasts express larger quantities of DQ molecules than B lymphocytes and monocytes. The results confirm that complement fixation microtechnique against PHA blasts is efficient for HLA-DQw typing.     

Isolation and characterization of the cDNA clone and genomic clones of a new HLA class II antigen heavy chain, DO alpha

From a human cDNA library constructed from a consanguineous HLA-homozygous cell line, AKIBA (HLA-A24, Bw52, DR2, Dw12, DQw1, and Cp63) (Cp63, a new SB type), a cDNA clone encoding a new HLA class II antigen heavy chain named DQ alpha was isolated, and was analyzed by Southern blot hybridization and by nucleotide sequence determination. The nucleotide sequence of the DO alpha cDNA was distinct from those of the DR alpha, the DQ alpha, and the DP alpha cDNA, but showed some characteristic features of the class II antigen alpha-chains. We also isolated and identified genomic clones specifying the DO alpha gene. Genomic analyses of cell lines with different HLA-DR serotypes with the use of the DO alpha cDNA as a probe indicated the existence of a single DO alpha gene that exhibited little restriction enzyme polymorphism.     

Molecular Diversity of HLA-DQ

HLA-DQ molecules were isolated from a panel of HLA-DR-DQ homozygous cell lines, partially of consanguineous origin, derived by the use of monoclonal antibody SPV-L3, and subsequently analyzed by gel electrophoretic techniques. It is demonstrated that both the DQ alpha and beta chain exhibit an extensive isoelectric point polymorphism. Within a panel of 29 B-cell lines tested, at least 5 distinct alpha and 6 distinct DQ beta chain gene products were observed. Of the 30 theoretically possible DQ alpha-beta dimers, only 10 could be identified within the panel: 5 different dimers are associated with the DQw1 allospecificity; HLA-DQw2 and -DQw3 are associated with 2 types of dimers, whereas another DQ alpha-beta combination was expressed by a cell line with a so-called DQ-blank specificity. The relation between the specificities 2B3 and TA10 appeared to be complicated as far as DQ beta chain isoelectric point differences are concerned: monoclonal antibody IIB3 seems to be reactive with four distinct DQ beta chain alleles whereas monoclonal antibody TA10 only reacted with one type of DQ beta chain. These results suggest that the polymorphic DQ alpha and beta chains may both contribute to the definition of the HLA-DQ allospecificity. A particular DQ beta chain was present in two types of HLA-DQw1 molecules, as well as in one type of HLA-DQw2 and -DQw3 (2133 positive) molecule, and formed dimers with electrophoretic distinct DQ alpha chains. On the other hand, HLA-DQw2 molecules isolated from HLA-DR3-positive cells and one type of HLA-DQw3 (TA10 positive) molecule were found to be constructed of identical alpha chains but appeared to differ in the composition of their DQ beta chain gene products. The implications of these findings will be discussed.     

Molecular mapping of class II polymorphisms in the human major histocompatibility complex. I. DR beta

We have studied 27 cell lines homozygous by consanguinity for the major histocompatibility complex to establish the restriction fragment length polymorphism (RFLP) patterns seen with six different restriction enzymes (Bam HI, Bg1 II, Eco RI, Hinc II, Hind III, Pvu II) and DR beta chain probes. The probes used were a full-length cDNA DR beta probe and a probe specific for the 3' untranslated region. The RFLP obtained represent the first standard patterns for the individual haplotypes DR1 through 7 and DR9 as defined by genetically homozygous lines. The patterns obtained reflect the DR specificities closely, as well as the DRw52 and DRw53 specificities. These latter specificities are associated with the most prominent patterns of RFLP. Bands are present which are unique for the haplotypes DR1, DR2, DR4, DR7, DRw52, and DRw53, and could be used for typing these haplotypes in heterozygotes. Subtypes can be identified for all of the haplotypes except DR1. These subtypes indicate that there is an extensive amount of polymorphism in the DR subregion that has not been identified serologically.