M. Lau

Nomenclature for factors of the HLA system, 2010

The WHO Nomenclature Committee for Factors of the HLA System met following the 14th International HLA and Immunogenetics Workshop in Melbourne, Australia in December 2005 and Buzios, Brazil during the 15th International HLA and Immunogenetics Workshop in September 2008. This report documents the additions and revisions to the nomenclature of HLA specificities following the principles established in previous reports (1–18).

Comprehensive method for the typing of HLA-A, B, and C alleles by direct sequencing of PCR products obtained from genomic DNA

Molecular testing is gradually replacing standard typing techniques in the field of HLA because it allows higher resolution, which has significant functional implications. Although several techniques have been so far described for this purpose, the definitive means to determine which alleles are present in a particular sample is to identify their sequence. We describe a simplified method for typing HLA-A, B, and C alleles by direct sequencing of polymerase chain reaction (PCR) products amplified from genomic DNA that could allow large-scale handling of samples for clinical use. The template is the product of a nested PCR. A first round of PCR amplifications from genomic DNA is performed with three different sets of primers, one pair specific for each locus. The PCR products encompass exons 2 and 3, the regions of interest to determine the allele present. These fragments are a mixture of both alleles present in one locus. In a second round of PCRs using the first fragment as template, exons 2 and 3 are separately amplified and simultaneously tailed with sequences corresponding to fluorescent-labeled commercial primers. The sense and antisense sequence of each exon is obtained and compared with a database of all known HLA-A, B, or C alleles. Heterozygous positions are determined and the most probable alleles assigned. This simplified procedure has the practical advantage of allowing high-resolution typing of clinical material by utilizing the same genomic DNA used for standard molecular typing of HLA class I.

An update to HLA Nomenclature, 2010

The WHO Nomenclature Committee for Factors of the HLA System met during the 15th International Histocompatibility and Immunogenetics Workshop in Buzios, Brazil in September 2008. This update is an extract of the main report that documents the additions and revisions to the nomenclature of human leukocyte antigen (HLA) specificities following the principles established in previous reports.

A new HLA-C allele, C*08:43, identified during a UCLA Immunogenetics Centre cell exchange.

HLA-C*08:43 differs from HLA-C*08:02:01 by one nucleotide (A>G) at position 584 resulting in an amino acid change of 171 tyrosine to 171 cysteine.

Polymorphism at codons 114, 116, 145, and 163 muddle the typing of HLA-B*1304.

Genetic exchanges often muddle the typing of HLA class I molecules, this is also the case for HLA-B*1304. Serologic and molecular DNA class I typing report a B15/B55 type for cell 847, whereas DNA sequencing finds B*5501/B*1304. HLA-B*1304 differs by no more than four amino acids from other HLA-B13 molecules, a comparative analysis of the B13 and B15 families was therefore performed to determine why serologic and molecular DNA approaches report a B15 type for B*1304. Comparisons demonstrate that limited differences individuate the B15 and B13 molecules such that the genetic recombination of codons 145 and 163 in the class I heavy chains alpha 2 alpha helix prompt B*1304 to exhibit a B15X21 pattern of serologic cross-reactivity. Molecular DNA class I typing approaches are also swayed by genetic recombinations to type B*1304 as a B15 molecule: B15-like nucleotide sequences encoding residues 114, 116, and 145, lead B*1304 to exhibit a B15 PCR amplification pattern. Thus, genetic exchanges encoding key amino acids in the class I heavy chain lead molecular and serologic typing approaches to categorize HLA-B*1304 as a member of the B15 family.

Molecular characterization of HLA-A*6803.

More than 200HLA class I and class II typing laboratories participate in the International Cell Exchange (ICE). Laboratories participating in the typing of HLA class I molecules as part of the exchange are sent four unknown samples each month upon which class I typing is completed. The participating laboratories then communicate the class I type identified, the data from all participants is combined, and a report assessing class I typing consistency from laboratory to laboratory is published. Laboratories participating in the ICE have historically determined a class I type using alloantibodies: however, the realization that serologic typing cannot discriminate among all class I subtypes has led to the implementation of molecular class I HLA typing methods. In the autumn of 1995, serologic typing indicated that cell 859 expressedHLA-A32 and -A68 [with no allelic subtypes assigned; Fig. 1 (Lau 1995)], while the consensus HLA-A type assigned by the various DNA-based methods was A*3201/A*68012. Because two of 14 DNA labs assigned anA68variant and four DNA labs failed to assign a type other thanA*3201 (data not shown), we cloned and sequenced the two HLA-A alleles from cell 859 under the hypothesis that a newHLA-Avariant was present in cell 859 (Domena et al. 1993; Ellexson 1996). Indeed, a new HLAA68 allele elusive to both serologic and DNA typing techniques was identified. A similarity search found this new HLA-A68 allele to be most homologous to A*68012, from which it differs at a single nucleotide (C instead of G) at position 282. In accordance with this high level of similarity to other A68 alleles, the WHO nomenclature committee (Bodmer et al. 1995) assigned the name A*6803 to the newHLA-A allele. The single nucleotide substitution which differentiates A*6803 from A*68012is a coding substitution; A*6803has a histidine at position 70 in theα1 domain of the class I heavy chain, whileA*68012 has a glutamine (Table 1). Because the side chain of amino acid 70 is positioned to affect the stereochemistry of specificity pockets B and C (Saper et al. 1991), the substitution of a positively charged histidine (A*6803) for an uncharged glutamine (all other A68s) may alter the peptides bound by these A68 molecules; no other pair of class I molecules differs only at amino acid 70 (Parham 1995), such that comparisons of peptides eluted fromA*68012 and A*6803 will help to elucidate the effect of polymorphisms at amino acid 70.