Genotype analysis
The sample 17ZZ2298 was firstly subjected to high-resolution typing of HLA-A, B, C, DRB1 and DQB1 by BGI Next Generation Sequencing Typing method-RCHSBT [7]. Exon 1-7 of HLA-A, B and C, exon 1-3 of HLA-DRB1, and exon 2-3 of HLA-DQB1 were sequenced. The high-resolution HLA assignment of the sample was as follows: A*01:01:01, 11:01:01, B*15:32:01, 57:01:01, C*06:02:01, 12:03:01, DRB1*07:01:01, 12:01:01, DQB1*03:01:01, 03:03:02; however, when the sample was further analyzed by Sanger’s sequencing method using three different reagents (CSTB, Biocapital and GenDx) the assignment for HLA-A was A*01:01/11:126. The sample was then reanalyzed by the SSO method and Miseq sequencing-based typing (Onelambda). The assignment determined by these methods was HLA-A*01:01/11:01 and HLA-A*01:01/11:126, respectively. However, the Miseq assignment had the following system comments: “Warning: mismatch in an intron, Two or more variants cannot be phased.” Indicating the possibility that HLA-A*01:01/11:126 was not the correct assignment. The only difference between HLA-A*11:01:01:01 and HLA-A*11:126 was at c.874A>G in exon 4 (Fig. 1A).
The sample was then further analyzed by Miseq-based typing (GenDx). The data showed that there was a new allele, but exon 3 and exon 4 could not be phased with the MiSeq data. Therefore, the Miseq reads were further analyzed together with a low number of MiniON reads. The recommended genotype was HLA-A*01:01:01:01/A*11:126 (Fig. 1B); however, there were numerous mismatches between exon 4 and exon 6. All mismatches (indicated by blue or red triangles) were in the HLA-A*11 allele, and were located between the last heterozygous position in exon 4 (gDNA 1824) and intron 5 (gDNA 2437), which was heterozygous in this sample. The bases found at gDNA 1824 matched with the two reported HLA-A alleles. The first mismatched position in intron 4 (gDNA 1887) was heterozygous AC in this sample. All known HLA-A alleles have an A at this position. Thanks to the phasing information, we found that the C belongs to HLA-A*11new. The last two heterozygous positions (gDNA 2431 and gDNA 2437) have A-A in one allele and G-T in the other allele. A-A occurs in many HLA-A alleles while G-T is not present in any HLA-A alleles. When region 1887 – 2437 (matching with HLA-A*11:126) or region 1824 – 2437 (matching with HLA-A*11:01:01:01) were excluded, the data were an exact match with HLA-A. The typing results of HLA-A with each reagent and the final nomenclature are listed in Table 1.
Sequence blast and mutation analysis
The sequence of region 1824 – 2437 (612bp, because of an “AT” deletion in intron 5) was then blasted in the IMGT/HLA database. The results showed that 612/612 (100%) was an exact match with HLA-H*02:07/14/18, as shown in Table 2 (https://www.ebi.ac.uk/Tools/services/web_ncbiblast/toolresult.ebi?
jobId=ncbiblast-E20201104-031132-0326-63995603-p1m). It was suggested that the sample contained a new HLA-A*11 allele (HLA-A*11:335), which was the result of the interlocus genomic exchange of HLA-A*11:01:01:01 and HLA-H*02:07/14/18. The distance between HLA-H and HLA-A on chromosome 6 was approximately 50kb. Austin L. Hughes also indicated that interlocus recombination had been a recurrent feature in the evolutionary history of the HLA class I region and suggested that class I pseudogenes had arisen through the duplication of class I genes over a long period of time[8]. The alignment of the genomic sequence of HLA-A*11:01:01:01 with A*11:126, A*11:335, H*02:07, H*02:14 and H*02:18 is shown in Fig. 2.
Transmembrane property analysis
As shown in Table 3, the sequence of HLA-A*11:335 differs from HLA-A*11:01:01:01 by 10 nucleotide substitutions, which resulted in 3 synonymous mutations and 6 missense mutations, mainly in exon 5. Exon 5 encodes the transmembrane domain of HLA-A. We analyzed the effects of the 6 missense mutations on the property of the transmembrane domain using the PSIPRED online tool (http://bioinf.cs.ucl.ac.uk/psipred/). The results showed that although 6 missense (3 in the transmembrane domain) mutations were produced as a result of interlocus recombination between HLA-A and HLA-H, these mutations did not lead to destructive effect on the helix structure of the transmembrane domain (Fig. 3).
New allele nomenclature
The nucleotide sequence of the new allele had been submitted to the DNA Data Bank of Japan (Accession No. LC474859) and to the IPD-IMGT/HLA Database [5] (Submission No. HWS10054755). The name HLA-A*11:335 was officially assigned by the WHO Nomenclature Committee in May 2019. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report [9], names will be assigned to new sequences as they are identified. The lists of these new names will be published in the next WHO Nomenclature Report.