A novel method for simultaneous high resolution identification of HLA-A, HLA-B, and HLA-Cw alleles.

Abstract

We describe a novel high resolution DNA based typing approach for HLA class I alleles, which identifies the recombinational motifs present in exons 2 and 3 of the HLA class I genes. Unique identification patterns for 201 known HLA-A, HLA-B, and HLA-Cw alleles were generated by the use of only 40 probes, which were targeted at these common motifs. The unambiguous identification of the alleles was achieved by the development of a new and powerful allelic separation technique that allows isolation of single alleles after amplification. To validate the method, we have used locus-specific primers to amplify exons 2 and 3 of HLA-A, HLA-B, and HLA-Cw loci from 22 heterozygous and 41 homozygous cell lines. After amplification, the allelic fragments from each locus were separated, blotted, and hybridized with the 40 probes. In all cases, the allelic products could be separated and 81 different class I alleles, 33 HLA-A, 30 HLA-B, and 18 HLA-Cw, were identified according to the predicted probe hybridization patterns.HLA-A, HLA-B, and HLA-Cw antigens are polymorphic cell surface glycoproteins, which are involved in cytotoxic T-cell recognition during the immune response. HLA matching is essential for kidney and bone marrow transplantation (1-4) and new molecular biological methods (5) for detection of HLA polymorphism currently provide an opportunity to improve matching of the patients and donors, as well as a research tool to investigate the relationship between HLA disparity and transplant complications. These molecular typing methods include sequence-specific primer (SSP) amplification (6, 7), hybridization with sequence-specific oligonucleotide (SSO) probes (8, 9), heteroduplex analysis (10-12), single-strand conformation polymorphism (13, 14), and direct nucleotide sequencing (14,15). SSP uses group and/or allele specific sequences for primer design (7) and has been proposed as a single technique for HLA class I and II typing (16); SSO is based on the identification of allele-specific sequences, which are either unique or in certain combinations will allow the identification of the allele (9). The resolution of each method could be increased by using a larger number of primer mixes for SSP and more probes in the case of SSO. These two methods have also been used for class I typing, but their application to high-resolution class I allele analysis has been hindered due to the extensive sequence homology between the HLA-A, -B and -Cw loci (17), between class I classical and nonclassical genes and the reported pseudogenes (18). Direct sequencing is at present used for high-resolution analysis of the class I alleles (14, 15) and, to avoid ambiguous combinations, prior typing is required, which is then followed by allelespecific PCR amplification to generate single allelic fragments. Although this approach achieves the highest resolution, it is expensive and difficult to perform routinely. Heterozygous combinations of certain alleles frequently produce ambiguous

Novel classical MHC class I alleles identified in horses by sequencing clones of reverse transcription‐PCR products

“…Previous studies of human MHC class I genes revealed locus‐specific sequences in introns which were used to make differential primers to PCR‐amplify HLA‐A, ‐B and ‐C genes (Cereb et al ., 1995; Arguello et al ., 1996; de Groot et al ., 2000; Johnson et al ., 2000). Whether the introns of horse classical MHC class I loci are locus‐specific is not known.…”

“…The α‐1 and α‐2 domains in classical MHC class I genes contain the peptide binding regions (PBR) where polymorphism most frequently occurs, while the TM and cytoplasmic domains are relatively conserved among classical MHC class I genes in horses (Holmes & Ellis, 1999) and humans (Kaufman et al ., 1994; Parham & Ohta, 1996). Analysis of intron sequences of human MHC class I genes revealed locus‐specific regions, which were used to make primers which differentially amplified HLA‐A, ‐B and ‐C genes by PCR (Cereb et al ., 1995, 1997; Arguello et al ., 1996; de Groot et al ., 2000; Johnson et al ., 2000). Although 13 horse MHC class I cDNA sequences, including four putative non‐classical MHC class I gene sequences, have been published (Barbis et al ., 1994; Ellis et al ., 1995; Holmes & Ellis, 1999; McGuire et al ., 2003), locus‐specific sequences were not found in those exon sequences.…”

Molecular matching in allogeneic bone marrow transplantation

“…Identification of the DNA sequence of a single HLA allele gives an unambiguous type. However, as two alleles at a heterozygous locus have to be analysed, sequencing both accurately requires separation by cloning (Prokupek et al , 1998) or strand separation (Arguello et al , 1996), making such an approach for routine typing impractical. Direct sequencing of both HLA alleles together can be achieved, and with an automated DNA sequencer together with specific HLA ‘sequence‐based typing software’, this approach becomes more applicable for use in the routine tissue‐typing laboratory (Petersdorf & Hansen, 1995; Johnston‐Dow et al , 1997; Scheltinga et al , 1997).…”

Physical separation of HLA‐A alleles by denaturing high‐performance liquid chromatography

“…Physical separation of alleles may be achieved by group-or sequence-specific PCR (1), or by various methods based on allelic differences in DNA conformation (2)(3)(4)(5)(6). Allele separation by groupspecific amplification reduces the complexity of the subsequent genomic typing.…”