An improved and validated RNA HLA class I SBT approach for obtaining full length coding sequences.

The functional relevance of human leukocyte antigen (HLA) class I allele polymorphism beyond exons 2 and 3 is difficult to address because more than 70% of the HLA class I alleles are defined by exons 2 and 3 sequences only. For routine application on clinical samples we improved and validated the HLA sequence-based typing (SBT) approach based on RNA templates, using either a single locus-specific or two overlapping group-specific polymerase chain reaction (PCR) amplifications, with three forward and three reverse sequencing reactions for full length sequencing. Locus-specific HLA typing with RNA SBT of a reference panel, representing the major antigen groups, showed identical results compared to DNA SBT typing. Alleles encountered with unknown exons in the IMGT/HLA database and three samples, two with Null and one with a Low expressed allele, have been addressed by the group-specific RNA SBT approach to obtain full length coding sequences. This RNA SBT approach has proven its value in our routine full length definition of alleles.

New insights in HLA-E polymorphism by refined analysis of the full-length gene.

BACKGROUND: Human leukocyte antigen (HLA)-E is a non-classical HLA class I molecule that plays a role in both the innate and the adaptive immune response through interaction with receptors on natural killer- and T-cells. The HLA-E gene is characterized by limited polymorphism compared with the classical HLA loci on chromosome 6. At the start of this study, only 13 variable sites had been identified (IPD-IMGT/HLA Database v3.18.0). While most previous studies focused on polymorphism in exons 2 and 3 or specific gene regions, polymorphism in the other exons and introns could influence protein expression and function as well. Studies that investigate extended HLA-E polymorphism are therefore needed to better understand the functional relevance of HLA-E in health and disease. AIMS: The aim of this study was to examine the variability of the full-length HLA-E gene region in individuals originating from different populations. MATERIALS AND METHODS/RESULTS: A total of 7 new HLA-E alleles were identified using full-length HLA-E sequencing of 123 individuals from Asian, Dutch or Hunan Han origin. Furthermore, genome variation analysis of the third phase of the 1000 genomes database showed 107 new variable sites in 2504 individuals originating from 26 different populations. DISCUSSION AND CONCLUSION: Our study demonstrates that the nucleotide variability of the HLA-E gene is much higher than previously known, albeit in only a limited number of individuals. Overall only 2 variants, HLA-E*01:01 and *01:03, are frequently present worldwide, suggesting that balancing selection is acting on HLA-E.