(500) Characterization of a Novel HLA-DQB1*04 Allele with Intron 4 Splice Variant: Implications for Transplant Compatibility

Body: With the widespread use of Next-Generation Sequencing (NGS) for HLA genotyping, ~6,000 novel alleles are added annually to the IPD-IMGT/HLA database. Assessing the nature of variations, particularly non-synonymous ones, is crucial for evaluating HLA compatibility in transplantation, as they can alter antigen recognition or HLA protein expression.
We present a case of a 29-year-old female evaluated as a potential living liver donor for her son. HLA typing by NGS (Holotype HLA™; Omixon/Werfen) identified a novel HLA-DQB1*04 allele differing from the reference DQB1*04:02:01:04 by a single nucleotide G>A substitution in exon 5, encoding cytoplasmic domain. Recipient typing revealed he shared the novel HLA-DQB1*04 allele with the donor. A novel HLA variant in the cytoplasmic domain may affect surface expression, trafficking, and protein interactions, potentially altering allo-responses. To assess the impact of novelty, we used the built-in amino acid sequence analysis tool (HLATwin™ 4.9.0), which provided protein sequence data for all exons except exon 5, thus lacking clarity on the substitution's nature (Figure 1). In contrast, NGSengine®-Turbo (GenDx) confirmed a non-synonymous nature of G>A substitution, resulting in Gly229Ala (Figure 2). Further analysis revealed that the patient had an “A” nucleotide present at the acceptor splice site at the end of intron 4, which is known to splice out exon 5. This aligns with previous studies showing that HLA-DQB1 exon 5 expression varies based on a G/A polymorphism at the intron 4 splice site. When "A" is present, exon 5 is usually spliced out, producing a protein shorter by eight amino acids. This analysis confirmed that exon 5 may not be expressed in the mature DQ4 protein in the present case. Although the impact of exon 5 exclusion on HLA-DQ expression is unknown, the observed novelty appears clinically insignificant given its absence in the mature protein.

Conclusion: In this case, both the reference and novel DQB1*04:02 alleles carry a G>A substitution at the intron 4 splice site, leading to the exclusion of Exon 5 from the mature HLA-DQ4 protein. As the resulting amino acid change is not expressed, the variant is clinically insignificant for transplantation. This underscores the importance of assessing intronic splice sites and sequence-level data when evaluating the impact of novel HLA alleles.