Lara Hector (Birkenfeld / DE; Zweibrücken / DE), Yannik Busch (Birkenfeld / DE), Tanja Brigadski (Zweibrücken / DE), Oliver Müller (Zweibrücken / DE), Marco Schäfer (Birkenfeld / DE), Wolfgang Peter (Birkenfeld / DE; Köln / DE)
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no conflict of interest
Compared to the classical HLA genes known to be relevant for transplantation, there is limited data on HLA-DOB. HLA-DOB codes for the β-chain of the HLA-DO class II molecule, which influences the peptide loading of classical HLA class II molecules. Due to limited research on this locus, it was assumed earlier that HLA-DOB has a low degree of polymorphism.
We disproved this hypothesis by developing a long-range PCR assay for HLA-DOB and typing of nearly 500 randomly selected Western European samples with next generation sequencing according to Illumina and the NGSengine. We extensively tested the assay"s reliability and robustness, making it suitable for detailed research into this locus. It enables full-length sequencing of the HLA-DOB, including a large part of the untranslated regions, generating sequences with a length of approximately 9.2 kb. Any appearing unknown allele sequences were verified using nanopore sequencing by Oxford Nanopore Technologies.
We found that slightly more than half of the alleles analyzed corresponded to HLA-DOB*01:01:01. In addition, the sequenced region included an indel, whose variations were observed in specific alleles. We also identified single nucleotide polymorphisms apparently linked to the 1066 bp deletion found in most HLA-DOB alleles. No significant Linkage Disequilibrium between HLA-DOB and the loci HLA-DOA, -DMB and -DQB1 were observed. However, we discovered a new HLA-DOB allele with an exon mismatch causing an amino acid substitution. In addition, we submitted 19 further HLA-DOB sequences to the IPD-IMGT/HLA database, which contained intron mismatches or served as extensions of already listed alleles.
In summary, analyzing nearly 500 Western European samples uncovered new alleles, suggesting many more unknown sequences could be identified with a larger and more diverse sample cohort comprising different ethnicities. Our assay provides an excellent basis for further HLA-DOB research, including discovering new alleles and expanding statistical analyses and databases, making future HLA-DOB typing increasingly precise.