HLA‐B*44:138Q: Evidence for a confined deletion and recombination events in an otherwise unaffected HLA‐haplotype

We discovered a new HLA‐B allele, HLA‐B*44:138Q, and confirmed its segregation. For characterisation, we used serology, sequence specific oligonucleotide (SSO), sequence specific primer (SSP), and full length sequencing by Sanger and next‐generation sequencing. From an evolutionary point the 5′ part of the new allele is identical with alleles from the HLA‐B*44:02 group, while its 3′ part is identical to the HLA‐B*15:18:01:02 allele, the breakpoint being located somewhere between intron 3 and exon 4. The salient feature of the new allele is a deletion of codon 94 in exon 3, which is unique for HLA‐alleles reported so far. Gene conversion can be hypothesised in the generation of this HLA sequence; however, the deletion seems to have occurred additionally. Other HLA‐alleles of the new allele's haplotype were common alleles.

We discovered a new HLA-B allele, HLA-B*44:138Q, and confirmed its segregation. For characterisation, we used serology, sequence specific oligonucleotide (SSO), sequence specific primer (SSP), and full length sequencing by Sanger and next-generation sequencing. From an evolutionary point the 5 0 part of the new allele is identical with alleles from the HLA-B*44:02 group, while its 3 0 part is identical to the HLA-B*15:18:01:02 allele, the breakpoint being located somewhere between intron 3 and exon 4. The salient feature of the new allele is a deletion of codon 94 in exon 3, which is unique for HLA-alleles reported so far. Gene conversion can be hypothesised in the generation of this HLA sequence; however, the deletion seems to have occurred additionally. Other HLA-alleles of the new allele's haplotype were common alleles.
Low resolution HLA-typing of a patient indicated the existence of a novel allele. We proceeded by performing highresolution typing by sequencing. Because the characterisation of the new allele required full length sequence analysis, we performed Sanger sequencing on cloned long-range polymerase chain reaction (PCR) products or amplicons generated by allele specific primers. The advent of nextgeneration sequencing (NGS) allowed us to assess the comparability of both methods in respect to effort and reliability of data on this new sequence. Biochemical tests for expression of the new allele were impossible because of lack of material. However, serological analyses were applied to check the expression of HLA epitopes on the cell surface.
Both techniques, SSO and SSP, led to inconclusive results in the HLA-B typing of an human immunodeficiency virus-positive patient; the most probable genotype seemed HLA-B*07,*44. Subsequent Sanger sequencing of exons 1, 2, and 3 revealed a heterozygous genotype consisting of an HLA-B*07:02, and a novel HLA-B*44 related allele; thus, refining the initial findings. The sequence of the new allele could be perfectly aligned with HLA-B*44:02:01:01; there was a deletion of three bases at codon 94, although.
To exclude phasing artefacts, we extended the analyses by cloning and sequenced 20 clones spanning exons 1 to 3. By this strategy, the results from the heterozygous sequencing were confirmed.
To characterise the novel allele more comprehensively, we amplified the whole novel allele by long-range PCR using allele specific primers located in the 5 0 and 3 0 end of the gene. For Sanger sequence analyses, we used 11 sequencing primers (all oligonucleotides of this study are listed in Table 1) that created overlapping sequences of forward and reverse strands.
To further confirm these results, we performed fulllength sequencing of HLA-B alleles from the patient and all available family members by NGS. 8 This approach covered even longer regions of the 5'UTR and 3'UTR than we had obtained in the Sanger approach.
HLA-B*44:138Q characterised by recombination and unique deletion events. The name B*44:138Q has been officially assigned by the WHO Nomenclature Committee in October 2011. This follows the agreed policy that, subject to the conditions in the most recent Nomenclature Report, 14 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.  allele. In column "Designation" the names of the primers are listed; in column "Sequence (5 0 ->3 0 )" the sequence of the nucleotides is provided; in column "Coverage" the targeted gene and the location of the primers on this gene are indicated; in column "Reference" the origin of the primer sequence is listed: primers have been created by us ("in house") or have been taken from publications; in column "Sequence technology" the technique used is stated. Oligonucleotides marked with *are also used as sequencing primers. The primers have been positioned to allow a full coverage of the allele The family study confirmed the unequivocal segregation of the new allele ( Figure 1).
To further characterise the haplotypes, we typed other class I genes, HLA-A, C, E, and G and class II genes, HLA-DRB1, DRB3/4/5, DQA1, DQB1, DPA1, and DPB1; additionally the class I-related MICA genes were typed. All of those genes were typed at full length. The results are provided in Figure 1. All alleles observed have been listed in the database; it thus seems that the events, leading to the generation of the new HLA-B allele did not affect other functional genes of the haplotype.
Restricting the view on the sequence to exons 1 to 3, the relationship of the novel allele with the HLA-B*44 group seemed apparent: sequences were identical except the deletion of three nucleotides. After we had obtained the full length sequence, it became evident, that the region from the 5 0 end to Intron 3 matched perfectly with HLA-B*44:02:01:01 and other HLA-B*44 alleles. From position 1620 in exon 4, however, several mismatches appeared; this part of the sequence matched perfectly with HLA-B*15:18:01:02 (Figure 2), although. From position 1221 in intron 3 to position 1619 in exon 4 both putative founder alleles share the sequence, a recombination between the two alleles might therefore have occurred there. 9 By contrast, the deletion of three nucleotides in exon 3 is a unique feature of HLA-B*44:138Q, indicating an independent event, that led to the creation of this allele.
In this respect, the new allele differs from the majority of other HLA alleles, where a simple "fixation of a single recombination event was responsible for the origin" 9 ; whether these events have occurred simultaneously or in independent meioses remains impossible to decide.
Because of the fact that this deletion comprised three nucleotides and occurred within two identical neighbouring codons (ATC-ATC) the resulting protein was shortened by just one isoleucin. This deletion concerns the second domain at the edge between the α-helix and the β-sheet. It is not likely that this position is part of an epitope. 10,11 However, the conformation of the molecules might change which could involve modifications of serological epitopes. This assumption would be in concordance with results from lymphocytotoxic tests: sera specific for HLA-B44 (n = 2), HLA-B12 (n = 2), or Bw4 (n = 3) did not show any reactivities with cells of the HLA-B*44:138Q-positive individual.

Patient
Father FIGURE 1 Pedigree chart of the patient's family; A-D indicate HLA-haplotypes; only HLA-A, B, C, DRB1, and DQB1 genotypes at two field resolution are depicted for easy readability. The new allele is located on haplotype b, which is observed in the father and two siblings *the mother was at the time of typing already deceased, her haplotypes have been deduced. The typing has been performed by various methods: • Serology: we used 180 in house validated typing sera to detect HLA class I molecules on the cell surface.
• Low resolution typing: SSO typing was performed for HLA-A, -B, and -C genes using a commercial reverse dot blot assay (Dynal, Bloomsborough, UK), SSP typing was performed for the HLA B gene only using a commercial SSP assay (Genovision, Vienna, UK).
• Sequencing-based typing is described in Figure 2.  From a technical point, the analysis of the full-length gene by NGS was much simpler than the Sanger approach, because of the clonal nature of the NGS sequencing, there was no necessity to separate alleles beforehand and no additional sequencing primers had to be designed. The sequence of the HLA-B*44:138Q allele was concordant with the Sanger result except a homopolymer at position 3061 of the 3'UTR: With the Sanger technique, 3 cytosins were detected while the consensus of the NGS analysis indicated only 2 cytosins. Given that Sanger sequencing has represented gDNA the golden standard for HLA typing for decades 12 this discrepancy argues for a good concordance of the new technology, albeit we have compared only one of the several platforms.
In summary, the characterisation of HLA-B*44:138Q on the IonTorrent platform allowed a fast analysis of whole genes with much less effort compared with Sanger sequencing. However, the homopolymer issue 13 of NGS remains a diagnostic challenge. The generation of the new allele within a haplotype that otherwise consists of common HLA-alleles (as assessed by full length sequencing) indicates that the mechanisms leading to this new allele are restricted to a single gene.