Hemizygous amplification and complete Sanger sequencing of HLA-C*07:37:01:02 from a South European Caucasoid.

The novel C*07:37:01:02 was completely sequenced after haplo-specific amplification from a European Caucasoid carrying B*07:02.

Multiple strategies have been designed for high resolution HLA typing and characterization of newly discovered HLA alleles, owing to the intrinsic difficulty of isolating and accurately defining extremely polymorphic and highly homologous HLA genes. These features and the combinatorial nature of HLA polymorphism challenge the capacity for cis/trans phasing of allelic polymorphisms of even modern technologies, including simple next‐generation sequencing approaches. We and others have previously reported that the full genomic sequence of a given HLA class I allele can, in most instances, be routinely amplified in isolation from the accompanying allele.2, 3 This is achieved with primers targeting allelic polymorphisms in the 5′/3′‐untranslated regions (UTR), method designated allele Group‐Specific Amplification sequence‐based typing (GSA‐SBT) or allele‐specific hemizygous Sanger sequencing. Here, we use this strategy to characterize the complete genomic sequence of allele C*07:37:01:02, of which partial sequences had been obtained previously from Japanese individuals (Dr. Masahiro Satake lab, Tokyo Red Cross Blood Center, GenBank accession nos. AB248242 and LC361334, unpublished).

We tentatively identified C*07:37 using a probe‐hybridization method in the course of an external proficiency test (EPT). The sample, derived from a Caucasoid individual from Palencia (Spain), was provided, without previous HLA‐phenotype selection, by the EPT scheme organizer (GECLID, Garantía Externa de Calidad para Laboratorios de Inmunología Diagnóstica). To obtain allelic resolution, and, eventually, characterize the allele complete genomic sequence, GSA‐SBT was performed as previously published. In parallel, a novel forward primer (FHg‐196, 5′‐ggaggcgccgcgttgg‐3′), recognizing a distinctive polymorphism in the 5'‐UTR of C*07 and C*18, was substituted for FHc‐20. Sanger sequencing of the two amplicons with intronic primers in the core facility of our research institute yielded co‐incident results, and permitted the characterization of the HLA‐C genomic sequence (3266 bp), including exons 1‐8, the intervening introns, and part of the flanking UTR. The new sequence was submitted to the GenBank database (accession number: MG739377) and to the WHO Nomenclature Committee for Factors of the HLA System, which, following the rules established in the last Nomenclature report, designated it officially as HLA‐C*07:37 (C*07:37:01:01 in IPD‐IMGT/HLA v3.42). Unfortunately, the sequence originally deposited in GenBank and IPD‐IMGT/HLA v3.32 through v3.42 databases was found in October 2020 to contain two typographical errors (two adjacent C>T changes in intron 2); after correction, it was shown to be identical and was reassigned to a recently reported allele, C*07:37:01:02, as it will appear in the next database release.

The C*07:37:01:02 genomic sequence differs from that of C*07:02:01:03 at a single nucleotide (T>G at position 915, exon 3), which determines substitution of arginine for leucine 156. This residue is located in an alpha‐helix of the alpha‐2 domain, where it can interact with the T‐cell receptor, antigen peptides, and alloantibodies (Figure 1). Classical HLA class I molecules are highly polymorphic at position 156, where five different amino acids are commonly observed (aspartate, leucine, arginine, glutamine, and tryptophan). Within HLA‐C, arginine 156 is seen in the majority of C*01, *04, *05, *14, *18 alleles, while leucine is mainly found in C*03, *07, *08, *15, and *17. Arginine 156 of C*07:37 is therefore an exception among C*07 alleles.

FIGURE 1

An HLA‐C*07:37 model highlighting arginine 156. The latter residue was represented with PyMOL (DeLano Scientific, San Francisco, CA) within a crystal structure of C*07:02 (Protein Data Bank ID: 5VGE)

Unfortunately, family segregation of the donor was unfeasible. However, his complete HLA type (A*02:01P; B*07:02:01,*44:05:01; C*02:02:02G,*07:37:01:02; DRB1*15:01:01G,*16:01:01; DRB5*01:01:01G,*02:02:01; DQB1*05,*06; DPB1*02:01:02G,*04:01P) suggests that C*07:37:01:02 emerged from C*07:02 within the well‐conserved haplotype C*07:02‐B*07:02‐DRB1*15:01. This is in contrast with the Japanese reference donors, who seemed to share a C*07:37‐B*67:01 haplotype.

Polymorphism at position 156 of HLA class I molecules affects both peptide and heavy chain conformation, hence modifying peptide repertoire and T‐cell recognition. Therefore, position 156 is considered an important determinant of alloreactivity. In particular, HLA‐C leucine 156 vs arginine 156 mismatch was pointed out as a risk factor for severe acute graft vs host disease and malignancy relapse after hemopoietic stem cell transplant.

CONFLICTS OF INTEREST

The authors declare no conflict of interest.