Characterisation of RAET1E/ULBP4 exon 4 and 3' untranslated region genetic architecture reveals further diversity and allelic polymorphism.

NKG2D is a natural killer cell activating receptor recognising ligands on infected or tumorigenic cells, leading to their cytolysis. There are eight known genes encoding NKG2D ligands: MICA, MICB and ULBP1-6. MICA and MICB are highly polymorphic and well characterised, whilst ULBP ligands are less polymorphic and the functional implication of their diversity is not well understood. Using International HLA and Immunogenetics Workshop (IHIW) cell line DNA, we previously characterised alleles of the RAET1E gene (encoding ULBP4 proteins), including the 5' UTR promoter region and exons 1-3. We found 11 promoter haplotypes associating with alleles based on exons 1-3, revealing 19 alleles overall. The current study extends this analysis using 87 individual DNA samples from IHIW cell lines or cord blood to include RAET1E exon 4 and the 3' UTR, as polymorphism in these regions have not been previously investigated. We found two novel exon 4 polymorphisms encoding amino acid substitutions altering the transmembrane domain. An amino acid substitution at residue 233 was unique to the RAET1E*008 allele whereas the substitution at residue 237 was shared between groups of alleles. Additionally, four haplotypes were found based on 3' UTR sequences, which were unique to certain alleles or shared with allele groups based on exons 1-4 polymorphisms. Furthermore, putative microRNAs were identified that may interact with these polymorphic sites, repressing transcription and potentially affecting expression levels.

Fifty novel HLA-DPB1 alleles identified in a UK cohort of unrelated hematopoietic cell donors and recipients.

HLA-DPB1 is the classical HLA class II genes with the least recorded variation on the IPD-IMGT/HLA Database, suggesting the full extent of its diversity is perhaps yet to be characterized. Here, a full-gene typing strategy was employed to genotype a UK cohort of 1470 HCT recipients (n = 744) and donors (n = 726). In total, 2940 full-length HLA-DPB1 sequences were generated, comprising 193 distinct alleles. Of these, 107 sequences contained novel variation, totaling 49 unique intronic HLA-DPB1 alleles, and one coding variant (HLA-DPB1*1188:01). Full-gene sequencing resulted in zygosity changes for 129 individuals by identifying two distinct intronic variants of the same coding allele. We verified the existence of nine unconfirmed alleles and extended the sequence of two existing alleles on the IPD-IMGT/HLA Database.

Characterization of the novel HLA-DRB1*11:01:01:12N allele by sequencing-based typing.

HLA-DRB1*11:01:01:12N differs from HLA-DRB1*11:01:01:03 by one nucleotide substitution in intron 3 at position c.652+1G>C, hg19.

Widespread non-coding polymorphism in HLA class II genes of International HLA and Immunogenetics Workshop cell lines.

As the primary genetic determinant of immune recognition of self and non-self, the hyperpolymorphic HLA genes play key roles in disease association and transplantation. The large, variably sized HLA class II genes have historically been less well characterized than the shorter HLA class I genes. Here, we have used Pacific Biosciences Single Molecule Real-Time (SMRT®) DNA sequencing to perform four-field resolution HLA typing of HLA-DRB1/3/4/5, -DQA1, -DQB1, -DPA1 and -DPB1 from a panel of 181 B-lymphoblastoid cell lines from the International HLA and Immunogenetics Workshops. By interrogating all exons, introns, and the untranslated regions of these important reference cells, we have improved their HLA typing resolution on the IPD-IMGT/HLA database. We observed widespread non-coding polymorphism, with over twice as many unique genomic sequences identified compared with coding sequences (CDS). We submitted 263 unique sequences to the IPD-IMGT/HLA Database, often from multiple cell lines, including 114 confirmations of existing alleles, of which 30 were also extensions to full-length genomic sequences where only CDS was available previously. A total of 149 novel alleles were identified, largely differing from their closest reference allele sequences by a single nucleotide polymorphism (SNP). However, some highly divergent alleles were deemed to be recombinants, only detectable by full-length sequencing with long, phased reads. The fourth-field variation we observed allowed fine mapping of linkage disequilibrium patterns and haplotypes to particular ancestries. This study has highlighted the under-appreciated non-coding diversity in HLA class II genes, with potential implications for population genetic and clinical studies.

Characterization of the novel HLA-A*02:01:01:206 allele in a Northern European individual.

Pacific Biosciences SMRT sequencing used to identify the novel allele, HLA-A*02:01:02:206.

The novel HLA-DRB1*03:01:01:05 and -DPB1*04:02:01:21 alleles identified in patients with acute leukemia.

Two novel HLA class II alleles were characterized, HLA-DRB1*03:01:01:05 and HLA-DPB1*04:02:01:21.

Characterization of the novel HLA-DPB1*665:01:02 allele by sequencing-based typing.

HLA-DPB1*665:01:02 differs from HLA-DPB1*665:01:01 by one nucleotide substitution in codon 139 in exon 3.

HLA-DPB1*64:01N and DPB1*701:01 sequence extensions by single molecule real-time DNA sequencing.

Pacific Bioscience's SMRT sequencing was used to confirm and extend HLA-DPB1*64:01N and 701:01.

Two novel alleles, HLA-A*32:01:01:09 and 32:01:01:10, identified by Pacific Bioscience's SMRT sequencing.

Pacific Bioscience's SMRT DNA sequencing was used to identify two novel intronic variants of HLA-A*32:01:01:01.