M. G. J. Tilanus

Correlations between polymorphisms at the DNA and at the protein level of DRw52 haplotypes, revealed with a variety of techniques

LB-Q1 and LB-Q4 are two subtypes of DRw52, defined by proliferative T-cell clones. These subtypes represent a polymorphism of the DR beta III gene. Similar subtypes of DRw52 can be defined by oligonucleotide typing, serology and RFLP analysis. In the present study we compared these typing techniques on a panel of 22 HLA-D homozygous, DRw52-positive typing cells. All typing techniques correlated very well. Three subtypes of DRw52 could be identified. Our results show that typing for cellularly defined structures can be done with a variety of noncellular techniques. This observation has important implications for matching in unrelated bone marrow transplantation and for disease association studies.

Molecular localization of LB-Q1, a DRw52-like T-cell recognition epitope and identification at the genomic level of associated shared hybridizing fragments☆

In this paper we report on the molecular localization of LB-Q1, a supertypic HLA class II determinant which we previously identified by the use of proliferative T cells. The population distribution shows that each of the DRw52 associated specificities DR3, DR5, and DRw6 may occur with and without LB-Q1. DNA from nine DR3, six DR5, and 14 DRw6 homozygous B-cell lines were digested with the enzymes TaqI, EcoRI, and PvuII. Using a DR beta cDNA probe, shared hybridizing fragments were observed that correlate completely with the presence or absence of LB-Q1. T-cell recognition of LB-Q1 can be blocked with a monoclonal antibody (7.3.19.1) which in some haplotypes selectively reacts with the DR beta III chains, but cannot be blocked with a monoclonal antibody (I-LR2) reacting in those same haplotypes exclusively with DR beta I chains. Therefore, LB-Q1 maps to the DR beta III molecule. These data suggest the occurrence of relatively frequent previous recombinations between the two DR beta chain genes present in DRw52 haplotypes.

Polymorphism and complexity of HLA-DR: evidence for intra-HLA-DR region crossing-over events.

HLA-DR molecules were isolated from HLA-DR3, −5, and −w6 positive homozygous B-cell lines by immunoprecipitation with monoclonal antibodies and analyzed by gel electrophoretic techniques. DNA isolated from the same cell lines was digested with the restriction enzyme Taq I and hybridized with a DR beta full-length cDNA probe. We demonstrated that certain DRβI alleles are found in combination with different DRβIII alleles as defined by Southern blotting, protein chemistry, a functional assay using purified protein derivative-specific T-cell lines, and, in one case, also alloreactive T-cell reagents. Our results indicate that within the family of HLA-DRw52-associated haplotypes DR beta chain genes may have been transferred from one haplotype to another. The implications of these findings are discussed.

HLA class II DNA analysis by RFLP reveals novel class II polymorphism

Polymorphism of the HLA-D/DR region has been defined by serologic and cellular methods. Additionally, protein and DNA analyses not only confirmed and refined the definition of the established polymorphisms but also revealed further polymorphisms for which no serologic or cellular correlate are known (yet). To study these in more detail, we analyzed the banding patterns obtained from Southern blot hybridizations with DR beta and DQ alpha cDNA probes. Specific fragments reflecting already defined polymorphisms could be identified. A refined HLA-D/DR definition based upon the presence of DNA subtypes could be introduced. Fragments have also been identified that are associated with the DR/Dw specificities. Moreover, individuals with different DR types may also share fragments in hybridization assays. These shared hybridizing fragments (SHFs) are, for instance, found in individuals typed as DR4, DR5, DR7, and DRw8. In total, 20 SHFs were found using two restriction enzymes and the DR beta and DQ alpha cDNA probes. Some of these SHFs correlate with antigenic determinants defined by broad reacting alloantisera, such as DRw52, but for 14 of these SHFs, no serologic equivalent has been found so far. Thus, SHFs reflect a conservation at the DNA level of the HLA class II region, which suggests that the polymorphic class II genes may be more conserved than previously thought. The possible biologic implications of conserved sequences in the HLA class II genes will be discussed.

Polymorphisms within the HLA-DRw6 haplotype. III. DQα and DQβ polymorphism associated with HLA-D☆

Abstract We have studied HLA-DQ encoded antigens from HLA-DRw6 homozygous cells and analyzed the DQ region at the DNA level. HLA-DQ molecules were isolated from EBV transformed B-cell lines and analyzed for DQα and DQβ polymorphisms. From the same set of cells, DNA was isolated and analyzed for RFLP. Polymorphism could be detected by both techniques, i.e., on the protein level and on the DNA. The variation in pI of the DQα and β chains correlated with the polymorphism as detected by HTC typing, as did the variation in molecular weight of the bands hybridizing to DQ specific cDNA probes; identical patterns were detected for cells of one HLA-D specificity and different patterns for different HLA-D types. Additionally, DQ reactive PLT reagents were raised against DRw6 positive cells. Panel studies revealed that these DQ reactive proliferative T cells can discriminate between the polymorphic DQ antigens on cells with different HLA-D specificities.

Two newly identified HLA-DRB1 alleles: DRB1*1322 and DRB1*1327.

The HLA-DRBgroup of ‘DR52’ related alleles encode the DR3, DR5, DR6, and DR8 antigens. With molecular techniques almost 150 different DRB1 alleles and 11 DRB3 alleles can now be distinguished (Bodmer et al. 1997). An irregular typing was observed when a Dutch Caucasoid individual, GVDP, serologically characterized as A2/A24(9) B51(5)/B15 DR1/DR6 , typed asDRB1*01and DRB3-positive in routine DNA typing by sequence-specific priming (SSP) in a polymerase chain reaction (PCR) (Olerup et al. 1992). This suggested either a DRB1-blank or an unexpected polymorphism at the SSP primer annealing site. By Sequencing Based Typing (McGinnis et al. 1995) the new allele was directly identified as a variant at the 39 primer annealing site of the DR11SSP mix, since in SBT the primers are located at a different location. The new allele was confirmed by cloning and subsequent sequencing. For cloning, a separate PCR product was generated in which the 59 upstream primer is located in intron 1 (5 9 CCG GTC GAC TGT CCC CCC AGC ACG TTT C3 9) and a biotinylated 39 primer mainly located in intron 2 (5 9 ACA CGA ATT CCC GCG CCG CGC TCA CCT C3 9). The upstream primer was extended with the restriction enzyme site forSalI and the downstream primer with an EcoRI site (underlined in the sequence shown). The digestions were performed on PCR template prepared with the use of streptavidin-coated beads (Dynal, Oslo, Norway). After each digestion, first with the restriction enzyme Sal I and subsequently withEco RI, the PCR fragment was purified with the streptavidin-coated beads. The PCR fragment, in the supernatant, resulted in a size of approximately 300 base pairs (bp). This fragment was ligated in the multiple cloning site of the pUC19 vector and introduced into competentEscherichia coli JM109 . Small-scale plasmid isolation according to the CTAB method (del Sal et al. 1989) revealed the DNA template for automated sequenci g using an ABI 373 DNA sequencer (ABD, Foster City, CA). Distinct individual clones were sequenced in sense orientation using dye-labeled-21M13 primers as well as in reverse orientation using dye-labeled M13RP1 primers. The sequence data from the clones were identical to the direct sequence data and confirmed the presence of a new allele. The WHO Nomenclature Committee assigned this allele to theDRB1*13group, based upon the presence of an alanine residue at codon 58 and the official name became DRB1*1322. In Figure 1 the exon 2 nucleotide sequence of DRB1*1322 is represented withDRB1*1102and with DRB1*0101as the consensus sequences. The new allele DRB1*1322 differs at codon 58 from theDRB1*1102 allele. Using two different primers specific for codon 58 this new allele can be identified in PCR-SSP. The sample NVE (a healthy Dutch Caucasoid blood donor) was submitted to the 12th Allele and Haplotype Society #15 ( DR6) because an unusual DR13/DQ2haplotype was identified by serology. Its HLA phenotype is serologically characterized as A2/A3 B8/B44 Cw5/Cw7 DR4/DR13 DQ2/DQ7 . The high resolution class II typing results are: DRB1*0401/ *13xx DRB3*0101 DRB4*01 DQA1*0501/*03 DQB1*0201/*0301 DPB1*0101/*0401 . The 12th IHW PCR-sequence-specific oligoprobes (SSO) analysis of the sample NVE showed an unusual hybridization pattern The nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank nucleotide sequence databases and have been assigned the accession numbers X86326 (GVDP = HLADRB1*1322), X97601 (NVE =HLA-DRB1*1327). The names DRB1*1322andDRB1*1327(GVDP and NVE, respectively) were officially assigned by the WHO Nomenclature Committee in May 1995 and June 1996. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report, names will be assigned to new sequences as they are identified. Lists of such new names will be published in the following WHO Nomenclature Report

Subtypes of DRw52: Different Typing Techniques Reveal Very Similar but Distinct Typing Results

LB-Q1* is a cellularly defined subtype of DRw52. The presence of LB-Q1 can be determined with proliferative alloantigen specific T-cell lines or clones (1,2). We have identified the T-cell recognition epitope LB-Q4, a postulated allele of LB-Q1. Association studies between LB-Q and established HLA-DR antigens revealed that both antigens LB-Q1 and LB-Q4, could be present in combination with DR3, DR5, and DRw6, but not with DRw8.

Evidence for Intra HLA-DR and -DQ Subregion Crossing Over Events

The HLA-DR and -DQ region of 47 homozygous typing cell lines were analyzed by a variety of techniques such as restriction fragment length polymorphism analysis, gel electrophoretic screening of the gene products, and in some cases, by testing MHC class II restriction in antigen presentation assays (1,2). Among these 47 cell lines, 16 different HLA-DR-DQ haplotypes were observed. Their genetic make up has been summarized in Table 1. It is concluded that some cell lines that express the same type of DRβI allele may express different kinds of DRβIII alleles. For example, cell lines positive for the AVL and QBL DRw17-DQw2 haplotypes do share the same DRβI3 gene product but AVL or QBL-like cell lines express DRβIII3 and DRβIII5 gene products, respectively. This DRβIII5 gene product is often expressed by HLA-DR5 positive cell lines and is involved in the expression of the T-cell recognition epitope LB-Q1 (3,4).

HLA Class II Restriction Fragment Length Polymorphism (RFLP) with Regard to the Class II Antigens

Mutational events in the genome may result in the creation of new restriction sites. The restriction fragment length variation obtained in Southern blot analyses, however, includes polymorphism caused by nonprotein coding sequences. The question is to what extent RFLP determinations contribute to the identification of polymorphisms related to class II antigens will be discussed.