Polymorphism clustering of the 21.5 kb DPA-promoter-DPB region reveals novel extended full-length haplotypes.

DPB1 and DPA1 genes share the same promoter region. Single-nucleotide polymorphisms (SNPs) within the regulatory regions of DP have been reported. This study hypothesizes that by including the SNPs in the promoter region of DP, extended haplotypes are defined, and promoter polymorphism is more extensive than what is currently reported. To identify the SNPs in the region of interest, the DP region spanning 21.5 kb was amplified in three separate long-ranged polymerase chain reactions. A DNA panel consisting of 100 samples was selected to represent a broad range of DPB1 alleles. The panel was amplified and sequenced using a dual sequencing strategy. Binary alignment map (BAM) alignments were generated and the mapped sequence alignments were analyzed using Integrative Genomics Viewer. A total of 76 SNPs were identified, and SNPs were clustered into 12 SNP-linked haplotypes. Multiple sequence alignments of promoter sequences indicated four distinct lineages within the connective region (CR) between two genes. The relationship between DPA1, CR, DPB1, and amino acid motifs was found to be correlated with HV1 and HV6. Of the 12 promoter haplotypes, DPB1 alleles observed with ProDP-4 were in complete linkage with HV1/2/5/6, the rs9277534G SNP, and the highly immunogenic T-cell epitope group. Multiple extended haplotypes of different intronic subtypes of the same DPB1 alleles were also identified. This new view of the full DP haplotype shows the relation of polymorphism, genes, and alleles, and provides a basis for future functionality related nomenclature. The novel clustering of the DP-extended haplotype warrants future investigations of DP haplotype matching in the outcome of haematopoietic stem cell transplantation (HSCT).

Insights into the polymorphism in HLA-DRA and its evolutionary relationship with HLA haplotypes.

HLA-DRA encodes the alpha chain of the HLA-DR protein, one of the classical HLA class II molecules. Reported polymorphism within HLA-DRA is currently limited compared with other HLA genes, as only a single polymorphism encodes an amino acid difference in the translated protein. Since this SNP (rs7192, HLA00662.1:g.4276G>T p.Val217Leu) lies within exon 4, in the region encoding the cytoplasmic tail, the resulting protein is effectively monomorphic. For this reason, in-depth studies on HLA-DRA and its function have been limited. However, analysis of sequences from the 1000 Genomes Project and preliminary data from our lab reveals unrepresented polymorphism within HLA-DRA, suggesting a more complex role within the MHC than previously assumed. This study focuses on elucidating the extent of HLA-DRA polymorphism, and extending our understanding of the gene's role in HLA-DR~HLA-DQ haplotypes. Ninety-eight samples were sequenced for full-length HLA-DRA, and from this analysis, we identified 20 novel SNP positions in the intronic sequences within the 5711 bp region represented in IPD-IMGT/HLA. This polymorphism gives rise to at least 22 novel HLA-DRA alleles, and the patterns of intronic and 3' UTR polymorphism correspond to HLA-DRA~HLA-DRB345~HLA-DRB1~HLA-DQB1 haplotypes. The current understanding of the organization of the genes within the HLA-DR region assumes a single lineage for the HLA-DRA gene, as opposed to multiple gene lineages, such as in HLA-DRB. This study suggests that the intron and 3' UTR polymorphism of HLA-DRA indicates different lineages, and represents the HLA-DRA~HLA-DRB345~HLA-DRB1~HLA-DQB1 haplotypes.

Full-length extension of HLA allele sequences by HLA allele-specific hemizygous Sanger sequencing (SSBT).

The gold standard for typing at the allele level of the highly polymorphic Human Leucocyte Antigen (HLA) gene system is sequence based typing. Since sequencing strategies have mainly focused on identification of the peptide binding groove, full-length sequence information is lacking for >90% of the HLA alleles. One of the goals of the 17th IHIWS workshop is to establish full-length sequences for as many HLA alleles as possible. In our component "Extension of HLA sequences by full-length HLA allele-specific hemizygous Sanger sequencing" we have used full-length hemizygous Sanger Sequence Based Typing to achieve this goal. We selected samples of which full length sequences were not available in the IPD-IMGT/HLA database. In total we have generated the full-length sequences of 48 HLA-A, 45 -B and 31 -C alleles. For HLA-A extended alleles, 39/48 showed no intron differences compared to the first allele of the corresponding allele group, for HLA-B this was 26/45 and for HLA-C 20/31. Comparing the intron sequences to other alleles of the same allele group revealed that in 5/48 HLA-A, 16/45 HLA-B and 8/31 HLA-C alleles the intron sequence was identical to another allele of the same allele group. In the remaining 10 cases, the sequence either showed polymorphism at a conserved nucleotide or was the result of a gene conversion event. Elucidation of the full-length sequence gives insight in the polymorphic content of the alleles and facilitates the identification of its evolutionary origin.