Immunogenetics38: 460-461, 1993 111111111no- genea'cs © Springer-Verlag 1993 A study of DR2-LUM haplotype generation and the DRB6*0202 linkage to DRBl*1601 Alfredo Corell 1., Pilar Varela 1., Pablo Morales 1, Estela Paz-Artall, Jorge Martinez-Laso 1, J. Manuel Martln-Villal, Ernette du Toit2, Antonio Arnaiz-Villena I 1 INMUNOLOGIA,HospitalUniversitario"12 de Octubre",UniversidadComplutense,CarreteraAndaluci~i,28041 Madrid, Spain 2ProvincialLaboratory for Tissue Immunology,Cape Town,SouthAfrica ReceivedMay 26, 1993/Revised versionreceivedJune 17, 1993 The DR subregion of the HLA system contains a single HLA-DRA gene (coding for the a chain of the DR c~[3 heterodimer) and varies significantly in length among individuals. The number of DRB genes ranges from one (in DR8 haplotypes) to four (in DR4, DRT, and DR9 haplotypes; Corell et al. 1991). A novel class II locus within the DR region has recently been characterized: HLA-DRB6 (Corell et al. 1991; Figueroa et al. 1991); this locus is present in about 26% of human chromo- somes (i. e., DR1, DR2, and DRIO haplotypes) and in all apes tested (Figueroa et al. 1991; Corell et al. 1992). Three DRB6 alleles have also been identified (Corell et al. 1992). Two novel DR1-DR2 hybrid haplotypes have also been described: one of them (designated DR1~2) has been characterized by using DNA Restriction fragment length polymorphism (RFLP) and sequencing (Bidwell et al. 1992), while the other (designated DR2-LUM) has been defined at the serological and RFLP level (Ouds- hoorn et al. 1990; Young et al. 1991). In the present work, the structure and origin of the DR2-LUM haplotype is put forward, based on the pres- ence of a particular DRB6 DNA allelic sequence ('0201); in addition, the absolute association between DRB6*0201 and DRB1 "1501 and between DRB6*0202 and DRB1 "1601 is established. Four Epstein-Barr virus-transformed cell lines: BRO-2 (DR "Br", DRIO), HOM-2 (DR1), HOM-1 The nucleotidesequencedata reported in this paper have been sub- mitted to the GenBank and EMBL nucleofide sequence databases and have been assigned the following accession numbers: EMBL- X53 357 (DRB6*0101), EMBL-X53358 (DRB6*0201), and Gen- Bank-M83204 (DRB6*0202). * The contributionto this paper by A. Corell and P. Varela is equal, and the order of authorshipis arbitrary. Correspondence to: A. Corell. (DR2), RML (DR16), and leukocytes from three in- dividuals [CGG (DR15), BA (DR1~2), DR2-LUM (Oudshoom et al. 1990)] were used for the complete DRB6 exon 2 sequencing by PCR amplification with generic DRB complete exon 2 primers (Corell et al. 1991), followed by M13 cloning. In additon, DNA from twenty-five HLA-DR2-posifive unrelated in- dividuals were used for the direct sequencing of specif- ic DRB6 partial exon 2 amplifications (Table 1). DRB gene exon 2 sequences obtained from the CGG and RML cell lines showed that the DRB6*0201 allele could be sprit into two different alleles differing only at one nucleotide position: the first base of codon 84 ("G" in DRB6-0201 and "A" in the new split DRB6-0202; Corell et al. 1992). This would originate a non-conser- vative Glycine-Arginine substitution, if expressed. Therefore, there are at least three different human HLA- DRB6 alleles (DRB6*0101, "0201, *0202). In order to obtain the distribution of these two new splits among different HLA-DR2 cells, a panel of 27 DR2-posifive cells (Table 1) was analyzed by specific DRB6 amplifi- cation (DRB-600 + DRB-AmpB primers) and direct DNA sequence analysis with the DRB-602 primer and dye-labeled dideoxy-terminators. All eighteen DR15 cells had the 84G allele (DRB6*0201), while all eight DR16 cells had the 841 allele (DRB6*0202). The cells with recombinant haplotypes (BA and DR2-LUM) con- tained the DRB6*0201 sequence. Thus, there was a 100% association between each DR2 subtype and the corresponding DRB6 allele in the non-recombinant ha- plotypes. DRB gene exon 2 sequences obtained from a DR2- LUM heterozygous individual showed only two differ- ent sequences, both belonging to the DR2 chromosome: 1) DRBI*1501 and 2) DRB6*0201. Of 50 clones sequenced, none were isolated that corresponded to any DRB5 allele. This suggests a lack of the DRB5 gene in the DR2-LUM haplotype, a notion further supported by