Identification of a new HLA-A allele (A*2502) by PCR-SSP

Abstract

A comprehensive polymerase chain reaction (PCR)-based system using sequence specific primers (SSP) now exists for tissue-typing, whereby HLA class I and II specificities can be simultaneously identified in one procedure (Bunce et al. 1995). This method, which determines HLA specificities based on their nucleotide sequence polymorphism, provides a more definitive approach to tissue-typing, offering easier discrimination and better resolution than serological evaluation. Because the method identifies sequence polymorphism, new specificities may present as anomalies in typing results. Using this system, we HLA-typed a Caucasian individual from genomic DNA. The result indicated a discrepancy at the HLA-A locus, with identification of three alleles (A*6601, A*2501, A*68). The rest of the genotype comprised B*0801, B*1402, Bw6; Cw*0701, Cw*0802; DRB1*0301/4, DRB1*1303, DRB3; DQB1*02, DQB1*0301/4. Both A*6601 and A*2501 belong to a broad serological specificity, HLA-A10. It was conceivable that the typing result represented a new sequence recombination involving elements of A*6601 and A*2501. The nature of the A*2502 sequence polymorphism was further determined through PCR gene-mapping (Krausa et al. 1995), whereby a panel of PCR-SSP reactions specifically identified the presence or absence of known sequence motifs in the appropriate region of the allele being characterized. This approach demonstrated that the new allele contains a combination of polymorphisms found in both A*2501 and A*6601. The variant resembles A*2501 in that it contains the characteristic Bw4 motif (Fig. 1, nucleotide position 300 –320), while it also contains a polymorphism at nucleotide position 282 found in A*6601 but not in A*2501. The combination of polymorphisms present within the A*2502 sequence explained the initial anomalous PCRSSP HLA typing result. The HLA-A*2502 gene was then cloned and sequenced, which involved cDNA synthesis through RT from total cellular mRNA, followed by the specific PCR amplification of full-length HLA-A locus genes. The HLA-A product was directly ligated into a pMOSblue T-vector (Amersham, Amersham, UK). Cells were then transformed and clones selected and screened by specific HLA-A PCR-SSP reactions (Krausa et al. 1996) to identify vectors containing HLA-A*2502. Four independent A*2502 clones were then sequenced on both strands. One clone was dideoxy sequenced using a standard approach (Sanger et al. 1977). Three other clones were full-length sequenced on both strands using the ABI377 DNA Sequencer technology (Perkin Elmer, Norwalk, CT). DNA sequencing verified the observations made in the initial PCR-SSP tissue-typing and gene-mapping. DNA sequencing of the A*2502 allele confirmed the polymorphism at nucleotide position 282, which distinguishes it from A*2501 (Fig. 1). As with many other HLA alleles, the nature of sequence polymorphism found within A*2502 probably arose through a gene conversion or recombination event (Parham et al. 1988), since the defining polymorphisms found within A*2502 are present in other HLA-A alleles. As with A*2501, this variant contains the Bw4 polymorphism. This is found between amino acid positions 77– 83, a region located at the top of the alpha helix of the alpha-1 domain of the class I molecule. Expression of the Bw4 motif has been shown to inhibit lysis by a subset of natural killer (NK) cells containing the NKB1 receptor (Gumperz et al. 1995). Although slightly different from HLA-B molecules containing the Bw4 motif, The nucleotide sequence data reported in this paper have been submitted to the GenBank/EMBL nucleotide sequence databases and have been assigned the accession number X97802. The name A*2502 was officially assigned by the WHO Nomenclature Committee in June 1996. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report (Bodmer et al. 1995), 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

DOI: 10.1007/s002510050175

Cite this paper

@article{Krausa1996IdentificationOA, title={Identification of a new HLA-A allele (A*2502) by PCR-SSP}, author={Peter Krausa and Dean M. Young and Frances Gotch}, journal={Immunogenetics}, year={1996}, volume={45}, pages={84-85} }