P016 Extending the IMGT/HLA database: Whole GENE sequencing of 247 distinct HLA-DPB1 alleles

Abstract

Aim Our current understanding of sequence variation in HLA-DPB1 is largely limited to exons 2 and 3, as only few alleles have been characterized at full length. To close these gaps in the IMGT/HLA database and to better understand patterns of polymorphisms in the HLA-DPB1 gene, we selected samples from the DKMS donor pool that represent all common and well-documented (CWD) alleles and performed phased full-length sequence characterization. In addition we characterized alleles that are currently not defined as CWD but occur at moderate to high frequencies in the DKMS donor populations. Methods We performed long-range PCR targeting the whole DPB1 gene using primers flanking the UTR-regions. The 12 kb amplicons were sequenced by two strategies on different platforms: Shotgun sequencing on Illumina MiSeq instruments and Single Molecule Real Time (SMRT) sequencing on PacBio® RS II instruments. Phase-defined allelic consensus sequences were generated from the long-read data and polished with the high-fidelity short reads using the DR2S software (DKMS Life Science Lab). Results We successfully sequenced the full-length DPB1 sequences of 331 samples (662 haplotypes) covering 40 common, 14 well-documented, and 40 not-CWD-defined alleles. In total, 247 distinct allelic variants were found considering also variation outside of exons 2 and 3. The 12 previously known full-length DPB1 alleles were each confirmed at least once. Most novel variation was found to reside in the intronic regions of DPB1. A phylogenetic analysis of the 247 alleles confirmed the existence of two major evolutionary branches characterized by two haplotype groups downstream of exon 2 with little internal variation and no evidence of historical recombination. These haplotype groups are tightly linked to the expression marker rs9277534 in the 3’ UTR. Conclusions We present the full-length sequences of 247 unique DPB1 alleles covering all common and all well-documented (CWD) variants. We have successfully submitted these alleles to IMGT/HLA, thus providing full genomic extension of 84 known DPB1 alleles and adding 151 novel DPB1 alleles to the database. Together, these alleles represent more than 99.9% of the DPB1 alleles encountered in the DKMS donor populations.