Jack Gorski

Structural comparison of the genes of two HLA-DR supertypic groups: the loci encoding DRw52 and DRw53 are not truly allelic.

The organization and sequence of the HLA-DRβ chain genes are compared in the two supertypic groups, DRw52 and DRw53, which together account for more than 80% of HLA-DR alleles. From the structural data, we conclude that these two groups represent distinct lineages which have followed different patterns of evolution. The fine structure of the β chain locus encoding the DRw53 specificity corresponds most closely to the DRβII pseudogene in the DRw52 haplotypes. Concomitantly, the DRβI locus in DRw53 haplotypes is more closely related to both of the two expressed DRβ loci of theDRw5 haplotypes (DRβI and DRβ III). These two loci are the result of a recent duplication. This leads to the proposal that both expressed DRβ chain genes in the DRw52 haplotypes (DRβI and DRβIII) are derived from a single precursor locus, while the two loci expressed in the DRw53 haplotypes are derived from distinct ancestral loci. The genes encoding DRw52 and DRw53 are therefore not true alleles of the same original locus. A scheme is proposed that accounts for the evolution of DR specificities within the DRw52 and DRw53 groups of haplotypes. It is evident that the differentHLA-DR alleles are not structurally equidistant and that one must take into consideration different degrees of heterozygosity or mismatch among the DR alleles.

DNA typing of DRw6 subtypes: Correlation with DRB1 and DRB3 allelic sequences by hybridization with oligonucleotide probes

Among MHC class II antigens, the DRw6/Dw6 complex represents a special situation where typing on a routine basis is often troublesome, mainly because monospecific alloantisera are rare and individual subtypes numerous. We demonstrate here that the use of oligonucleotide DNA typing permits an analysis of the polymorphism within DRw6 haplotypes and provides a molecular basis for correlations with functional data. Synthetic oligonucleotide probes, most of them locus- and allele-specific, were derived from the DNA sequences of three alleles of locus DRB1 and three alleles of locus DRB3. These probes allow the positive identification of distinct DRw6 subtypes. As analyzed on a panel of 26 well-defined DRw6 cell lines, oligotyping allows a direct and absolute correlation with the DRw13 serologic specificity and with the cellularly defined Dw9,Dw16,Dw18, and Dw19 specificities. Correlations of the polymorphism at the DRB1 locus with the polymorphism at the DRB3 locus (DRw52 alleles) allow us to identify preferential allelic associations such as DRw13-Dw18-DRw52a/52b, DRw13-Dw19-DRw52c, and DRw13/Dw19 haplotype, the Dw19 cellular reactivity might involve, at least DRw14-Dw9-DRw52b. In view of the absolute segregation of the DRw52c allele with the DRw13/Dw19 haplotype, the Dw19 cellular reactivity might involve, at least in part, epitopes on the DRw52c allele. The identification of DRw6 subtypes, as well as of other HLA class II subspecificities, by oligotyping can now complement and possibly replace serologic and cellular typing. It represents a particularly useful contribution to the optimization of class II matching in the case of bone marrow transplantation with unrelated donors.

Oligonucleotide genotyping shows that alleles at the HLA-DRβIII locus of the DRw52 supertypic group segregate independently of known DR or Dw specificities

Using locus- and allele-specific oligonucleotide probes, we have studied the polymorphism of the HLA-DRβIII locus within the haplotypes of the DRw52 supertypic group. DNA from a number of homozygous typing cells typed for both Dw and DR was used. The DRβIII polymorphisms, DRw52a and DRw52b, do not segregate with Dw typing, or with DR typing, indicating that the determinants responsible for Dw-defined T -cell response and for DR haplotypic recognition are not encoded by the DRβIII locus. Hence, we can conclude that these DR specificities are encoded by the other functional DR locus, DRβI, while the DRβIII locus encodes only the supertypic product.

Characterization of an HLA-DR Pseudogene in the DRw52 Supertypic Group

The nature of the DRβII pseudogene in a haplo-type of the DRw52 supertypic group was investigated by nucleotide sequence analysis. It revealed several deleterious mutations in the signal sequence and second domain regions in addition to the complete absence of the first domain and adjacent sequences. No expression of DRβII pseudogene mRNA can be detected. The same DRβII pseudogene is probably present in other members of the DRw52 supertypic group. The pattern of mutations in this DRβII pseudogene is different from that observed in the DRβ pseudogene of the DRw53 supertypic group, indicating a distinct evolutionary pathway for these two groups of DR haplotypes.

The single DR β gene of the DRw8 haplotype is closely related to the DR β 3III gene encoding DRw52

In most individuals two HLA-DRβ genes are expressed from each chromosome. One of these genes encodes one of the classical DR specificities, while the other encodes either of the supertypic DRw52/DRw53 specificities. In addition to these genes usually one or two DRβ pseudogenes are present. In contrast, the DRw8 chromosomal region only contains a single DRβ gene. To determine the relationship of this single gene to the multiple DRβ genes of other DR specificities, comparisons of Southern genomic blots were carried out. In this analysis genomic clones for each individual DRβ chain locus were included. The DRβw8 gene was indistinguishable from the DRβIII gene of DR3 cells (encoding DRw52), suggesting that it is closely related to the latter gene. The functional implications of this finding are discussed.

Molecular analysis of the genes for human class II antigens of the major histocompatibility complex

Different cDNA clones have been isolated that encode each of the three chains of HLA-DR antigens: alpha, intermediate and beta, as well as another beta chain, most likely DC. Whereas the DR alpha and intermediate chains seem encoded by single genes, the DR and DC beta chains are most likely encoded by multiple genes; furthermore, their polymorphism can be readily detected by restriction analysis of cellular DNA. Several genomic DNA clones were isolated for the DR and DC beta chain genes and for the intermediate chain gene. The sum of all distinct cDNA clones and genomic DNA clones for HLA-DR beta chains, isolated from a heterozygous cell line, represent five genes. This implies the existence of at least three nonallelic DR beta chain genes in addition to the DC beta chain genes. The complete sequence of one of the DR beta chains is presented. A genomic DNA clone for a DR beta chain was transferred into mouse L cells and found to be expressed into RNA of the same size as DR beta mRNA. The finding, among the genes for class II antigens, of multiple genes for the beta chain of HLA-DR, distinct from those of other known subregions such as DC, emphasizes the importance of gene transfer experiments, where individual genes can be expressed and tested for their functional role in the immune response.

Serological recognition of HLA-DR allodeterminant corresponding to DNA sequence involved in gene conversion

HLA class 11 molecules were isolated from mouse L cells transfected with a DRα gene and an allele, 52a, of locus DRβIII from an HLA-homozygous cell line, AVL, of the DR3 haplotype. The isolated molecules were found to possess a new allospecificity, named TR81. This specificity behaved allelic to the previously described DRβIII locus. The TR81 specificity was also present on the DRβI gene product of the DR3 haplotype. The nucleotide sequence of the gene encoding TR81 differs from TR81-negative DRβ genes of the DRw52 family in only two codons, both located in the regions known to be involved in a gene conversion event. Consequently, the following conclusions can be formulated. (a) TR81 is a bi-locus specificity and allelic to TR22 only in its DRβIII locus localization. (b) The TR81 specificity is the phenotypic counterpart of the gene conversion event which led to the generation of the DRβI gene of the DR3 haplotype. (c) One or both individual amino acid substitutions in the first domain of the DRβ chain are responsible for the TR81 allospecificity. (d) Since TR81 is expressed on the DRβI chain of the DR3 haplotype, it is possible that TR81 and DR3 represent the same serological specificity.

HLA-DRα, -DXα, and DRβIII gene association studies in DR3 individuals

Abstract In this study we have examined the results of probing with synthetic oligomers at the DRβIII locus, together with restriction fragment length polymorphisms defined by BglII digestion and a cDNA DRα probe, and Taq 1 digestion and a genomic DQα probe. We have demonstrated heterogeneity of the human leukocyte antigen DR3 and close association of the DRα, DRβIII, and DXα genes. Two DR3-related preferential allelic associations have been identified, which may prove useful in family analysis as well as for investigations of DR3-related diseases.

Oligonucleotide Typing Analysis of the Polymorphism of DRB1 and DRB3 Genes Within DRw52 Haplotypes

The HLA class II antigens are present as heterodimers of α and β chains on the surface of B cells, macrophages, and activated T cells (1). The polymorphism encoded in the class II DP, DQ, and DR subregions provide the structural basis for the immune responsiveness to foreign antigens. In particular the class II determinants play a key role in the MHC-restricted activation of T helper cells (2). The requirement for class II matching in transplantation and the HLA class II disease associations underline the medical importance of class II antigens. Cloning and sequencing data revealed that the genetic complexity of class II genes is greater than was expected from the serologic analyses (3,4). Using locus- and allele-specific oligonucleotide probes, we have been able to make true allelic comparisons of the two expressed DRβ chain loci, DRB1 and DRB3, formerly called DRβI and DRβIII (5,6), within a large panel of DRw52 individuals, including DR3, DR5, DRw6 and DRw8 haplotypes. Three alleles of DRw52 have been detected (7) and extended haplotypes were identified: A1,B8,DR3,52a; DR5(wll), 52b; DRw13,Dw19,52c and DRw14,Dw9,52b. Four splits of DRw6 corresponding to the Dw9, Dw16, Dw18 and Dw19 cellular specificities can now be discriminated by oligonucleotide typing.

Molecular Complexity of the HLA-DR, DQ, and DP Genes and Genotypic Split of HLA-DR Serological Specificities by “DNA Typing”

Class II or Ia antigens encoded within the D region of the major histocompatibility complex are composed of two transmembrane glycoproteins, the α chain (35 kd) and the β chain (29 kd). They are expressed on macrophages, certain lymphocytes, and some epithelial cells. The la molecules are characterized by extensive allelic polymorphism, which is responsible for the self-restriction of cell-cell interactions within the immune system. In the human MHC, several biochemically distinct groups of polymorphic class II antigens have been described: HLA-DR [1], DQ (DC) [2], and DP (SB) [3]. The functional properties of HLA class II molecules map in the D region of this complex. Several lines of evidence suggest that the biochemically defined surface antigens HLA-DR, DQ, and DP might be responsible for the functional properties of the D region [4, 5]. Our laboratory has exploited the method ology of gene cloning to isolate, identify, and study the genes coding for human class II antigens. We have concentrated on the study of β chain genes, which, in the case of HLA-DR, form the polymorphic component of greatest importance. The initial steps of that study have led to the cloning of HLA-DR and DQ β chain cDNA [6] and to the determination of the DNA sequence of a DR β chain cDNA [7]. These cDNA clones have then been used to identify the genomic genes for the HLA-DR, DQ, and DP β chains.