Characterisation of the Novel HLA‐B*51:01:01:126 Allele in a Greek Renal Transplant Candidate

ABSTRACT

The HLA‐B*51:01:01:126 allele differs from HLA‐B*51:01:01:01 by a single nucleotide substitution in intron 5.

The discovery and molecular characterization of previously unreported HLA alleles are fundamental to the ongoing elucidation of HLA genetic diversity. Novel HLA variants enrich our understanding of population‐specific allele distributions and provide valuable information regarding evolutionary history, immune‐mediated disease associations, and variability in therapeutic responses. Furthermore, the accurate identification and reporting of new alleles support the continuous refinement of the IPD‐IMGT/HLA Database and contribute to advances in histocompatibility assessment, transplantation medicine, and immunogenetics research [1, 2].

In this report a previously unreported HLA allele, now designated HLA‐B*51:01:01:126, is described. This allele was identified in a 58‐year‐old Greek male patient awaiting renal transplantation. The allele was detected during routine HLA typing for class I (HLA‐A, ‐B, ‐C) and class II (HLA‐DRB1, ‐DRB3/4/5, ‐DQA1, ‐DQB1, ‐DPA1, and ‐DPB1) loci by NGS technology, implemented at the Immunology and Histocompatibility Department at Evangelismos General Hospital in Athens, Greece.

The name HLA‐B*51:01:01:126 has been officially assigned by the World Health Organization (WHO) Nomenclature Committee for Factors of the HLA System in February 2025. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report [3], names will be assigned to new sequences as they are identified. Lists of such new names will be published in the following WHO Nomenclature Report.

Genomic DNA (gDNA) was isolated from peripheral blood using automatic extractor according to manufacturer's instructions (Maxwell Promega, Madison, Wisconsin). Gene amplification and library preparation were carried out using the commercial NGSgo‐MX11‐3 kit reagents (GenDx, Utrecht, the Netherlands), indexed with NGSgo‐IndX Plate I Kit (GenDx, Utrecht, the Netherlands) and quantified by the Qubit 1X dsDNA High‐Sensitivity Assay Kit (Thermo Fisher Scientific, MA, USA). NGS was implemented for full‐length genotyping of HLA‐A, ‐B, ‐C, ‐DQA1, ‐DQB1, ‐DPA1 (5′UTR–3′UTR), and partial sequencing of HLA‐DRB1, ‐DRB3/4/5, and ‐DPB1 loci in three separate PCR reactions per sample. Sequencing was performed on the Illumina MiSeq platform, (Illumina, San Diego, CA, USA) in both forward and reverse directions. Allele assignment and phasing were conducted by the NGSengine software v.3.2.0 (GenDx, Utrecht, the Netherlands) with references from the IPD‐IMGT/HLA Database v3.57.0. and custom parameters. All steps on MiSeq, including target generation, clonal amplification, library preparation and sequencing were performed according to the vendor and laboratory‐defined specifications [4].

The analysis software showed no perfect match with any known HLA‐B allele combination, suggesting the presence of a novel HLA‐B*51 variant. The full‐length DNA sequence of the novel HLA‐B*51:01:01:126 allele (GenBank accession number PV076211, IPD‐IMGT/HLA submission number HWS10100452) differs from the closely related HLA‐B*51:01:01:01 (IPD‐IMGT/HLA Acc No.: HLA00344) by one single nucleotide substitution (T>G), in intron 5, at gDNA position 2225. The difference is illustrated in Figure 1. The sample displayed a median depth of coverage of 530 reads at the HLA‐B locus, with the mismatched G base attributed to 307 reads at the specific position in the new HLA allele.

FIGURE 1.

Sequence alignment of the HLA‐B intron 5 sequence between the novel B*51:01:01:126 allele and the closely related B*51:01:01:01 allele. One single nucleotide substitution (T>G), in intron 5, at gDNA position 2225 was observed. Dashes (−) indicate identity between the B*51:01:01:01 allele. The numbers above the aligned sequence correspond to gDNA positions.

Confirmation of the presence of the novel HLA‐B*51:01:01:126 allele was performed by repeating HLA genotyping using different commercial locus‐specific primers supplied by CareDX (AlloSeq Tx17 kit). Complete gene sequencing was conducted for HLA‐A, ‐B, ‐C, ‐E, ‐F, ‐G, and ‐H, along with full exon sequencing for HLA‐DRB1, ‐DRB3/4/5, ‐DQA1, ‐DQB1, ‐DPA1, ‐DPB1, MICA and MICB. The raw sequencing data (FASTQ files) were analysed for base calling by AlloSeq Assign analysis software Tx17.1 v1.0.6 (CareDX, Stockholm, Sweden) with references from the IPD‐IMGT/HLA Database version 3.57.0. This nucleotide substitution was confirmed by the second procedure. The extended HLA genotyping for the patient was HLA‐A*03:01:01, 24:02:01; ‐C*02:02:02, 06:02:01; ‐B*13:02:01:01, 51:01:01:126; ‐DRB1*04:03:01G, 07:01:01G; ‐DRB4*01:01:01G; ‐DQA1*02:01:01G, 03:01:01G; ‐DQB1*02:01:01G, 03:01:01G; ‐DPA1*01:03:01G; ‐DPB1*04:01:01G.

Author Contributions

Diamanto Kouniaki performed the NGS sequencing, participated in the analyses of sequencing data and consensus sequences, contributed to the submission of the sequence data to GenBank and IPD‐IMGT/HLA Databases, as well as contributed to the writing and review of the manuscript; Sofia Nikolaou contributed to the review of the manuscript; Alexandra Tsirogianni directed the study and contributed to the review of the final manuscript. All authors have read and approved the final manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.