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HLA-C*15:255 differs from HLA-C*15:02:01 by five nucleotide substitutions located in exons 4 and 5., (© 2023 The Authors. HLA: Immune Response Genetics published by John Wiley & Sons Ltd.)

HLA-DPB1*1447:01 differs from HLA-DPB1*04:01:01 by a single nucleotide in exon 2 from A to C at position 134., (© 2023 The Authors. HLA: Immune Response Genetics published by John Wiley & Sons Ltd.)

HLA-C*16:201 differs from HLA-HLA-C*16:01:01 by one nucleotide substitution in codon 73 (ACT ->GCT)., (© 2023 The Authors. HLA: Immune Response Genetics published by John Wiley & Sons Ltd.)

HLA-B*35:574N contains a single nucleotide substitution at nucleotide position 2 (ATG to ACG)., (© 2023 The Authors. HLA: Immune Response Genetics published by John Wiley & Sons Ltd.)

HLA-C*07:1044N differs from HLA-C*07:01:01:01 by a single nucleotide deletion in exon 3, codon 124, nt. 442., (© 2023 The Authors. HLA: Immune Response Genetics published by John Wiley & Sons Ltd.)

HLA-A*03:420 differs from HLA-A*03:01:01:01 by a single nucleotide in exon 1, codon -22., (© 2022 The Authors. HLA: Immune Response Genetics published by John Wiley & Sons Ltd.)

HLA-A*03:57 has two amino acid exchanges in exon 2 at adjacent positions of the peptide-binding groove., (© 2011 John Wiley & Sons A/S.)

A novel human leukocyte antigen-DQB1 allele, DQB1*0323, was identified in a volunteer hematopoietic stem cell donor. DQB1*0323 differs from the closely related allele DQB1*030303 in five nucleotide positions.

Sequencing provides the most reliable and accurate information of the sequence of a gene and is therefore of particular interest for fully characterizing the genetic complexity and allelic diversity of human leukocyte antigens (HLA) genes. This complexity makes sequencing the method of choice for HLA low- and high-resolution typing. However, sequencing is considered a complex process, which requires the management of an overwhelming amount of data. However, only sequencing can be used as a sole typing technique without the need of supplementation by other typing techniques; it offers additional advantages by providing results in electronic formats and by being fully automatable. This makes sequencing the best candidate for bioinformatic and biomechanic integration to maintain the benefits and discard the drawbacks. We have, therefore, developed robotic platforms and a bioinformatic tool allowing complete documentation, interpretation and processing of all amplification and sequencing steps as well as the full integration of all instruments involved. It was shown that this integrative approach allows sequencing to run fully automated for low- and high-resolution typing on each throughput level desired. This integration of robotics and software secures the traceability of each specimen at each step of the process and minimizes the risk of contamination and sample mix-up.

Exon 3 of DRB1 is known to be polymorphic, but thought to be conserved within allelic groups. This implies that exon 3 polymorphisms would not need to be considered in evolutionary studies or clinical settings when assessing immunogenicity of allelic mismatches in stem cell transplantation. To further assess this, we determined the sequences of DRB1 exon 3 by hemizygote amplification and direct sequencing on 55 selected DNA samples containing 42 DRB1 alleles for which no exon 3 sequence data were previously available. The data confirmed the high degree of overall sequence conservation. The DRB4- and DRB5-associated alleles were completely conserved within their DRB1 groups. However, it could be shown that exon 3 is more diverse than previously expected. Multiple allelic differences within each group of DRB3-associated DRB1 alleles were found, without identifying unique group-related sequence motifs differentiating between these groups. For DRB1*1402 and DRB1*1406, it could be shown that they originated from DRB1*0302. In several samples previously typed as DRB1*1401, a novel DRB1 allele was identified: DRB1*1454. Thus, from a clinical viewpoint, the availability of exon 3 sequence information may be useful for optimizing typing as well as matching strategies. Additionally, it will allow for more detailed evolutionary studies, further elucidating the origin of alleles and the mechanisms driving sequence diversification.