The novel alleles, HLA-G*01:04:09 and HLA-DPB1*04:01:01:136, detected in a hematopoietic cell donor.

Two novel alleles, HLA-G*01:04:09 and HLA-DPB1*04:01:01:136, were identified in a single healthy individual.

The novel HLA-F*01:16 and HLA-F*01:17 alleles identified in hematopoietic cell donors.

Two novel non-classical HLA class I alleles have been characterized, HLA-F*01:16 and -F*01:17.

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.

86 novel HLA-E alleles discovered through full-gene sequencing of 6227 hematopoietic cell transplant patients and unrelated donors.

Until recently the number of alleles of the nonclassical HLA class I gene HLA-E documented in the IPD-IMGT/HLA Database was small and as a result, the gene was often not considered to be notably polymorphic. Here, we describe our work in identifying and submitting 86 novel HLA-E alleles after full-gene single-molecule real-time (SMRT) DNA sequencing of 6227 DNA samples. These samples were comprised of 2468 patients undergoing hematopoietic cell transplantation and 3759 unrelated potential donors. A total of 111 unique HLA-E alleles were detected in this cohort. The majority of novel alleles (79.1%) contained polymorphisms in intronic regions, highlighting the significant undiscovered variation present in the noncoding regions of the HLA-E gene.

Identification of the novel HLA-E*01:03:02:25 allele in an acute lymphoblastic leukemia patient.

HLA-E*01:03:02:25 sequenced from blood and buccal samples of an ALL patient.

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.

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.

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.

Impact of Previously Unrecognized HLA Mismatches Using Ultrahigh Resolution Typing in Unrelated Donor Hematopoietic Cell Transplantation.

PURPOSE: Ultrahigh resolution (UHR) HLA matching is reported to result in better outcomes following unrelated donor hematopoietic cell transplantation, improving survival and reducing post-transplant complications. However, most studies included relatively small numbers of patients. Here we report the findings from a large, multicenter validation study. METHODS: UHR HLA typing was available on 5,140 conventionally 10 out of 10 HLA-matched patients with malignant disease transplanted between 2008 and 2017. RESULTS: After UHR HLA typing, 82% of pairs remained 10 out of 10 UHR-matched; 12.3% of patients were 12 out of 12 UHR HLA-matched. Compared with 12 out of 12 UHR-matched patients, probabilities of grade 2-4 acute graft-versus-host disease (aGVHD) were significantly increased with UHR mismatches (overall P = .0019) and in those patients who were HLA-DPB1 T-cell epitope permissively mismatched or nonpermissively mismatched (overall P = .0011). In the T-cell-depleted subset, the degree of UHR HLA mismatch was only associated with increased transplant-related mortality (TRM) (overall P = .0068). In the T-cell-replete subset, UHR HLA matching was associated with a lower probability of aGVHD (overall P = .0020); 12 out of 12 UHR matching was associated with reduced TRM risk when compared with HLA-DPB1 T-cell epitope permissively mismatched patients, whereas nonpermissive mismatching resulted in a greater risk (overall P = .0003). CONCLUSION: This study did not confirm that UHR 12 out of 12 HLA matching increases the probability of overall survival but does demonstrate that aGVHD risk, and in certain settings TRM, is lowest in UHR HLA-matched pairs and thus warrants consideration when multiple 10 out of 10 HLA-matched donors of equivalent age are available.

The novel HLA-C*03:04:01:47 allele sequence identified using Pacific biosciences SMRT sequencing.

A novel variant of HLA-C*03:04:01:47 was identified using Pacific Biosciences SMRT sequencing platform.

Single molecule real-time DNA sequencing of the full HLA-E gene for 212 reference cell lines.

We have developed a genotyping assay that produces fully phased, unambiguous HLA-E genotyping using Pacific Biosciences' single molecule real-time DNA sequencing. In total 212 cell lines were genotyped, including the panel of 107 established at the 10th International Histocompatibility Workshop. Our results matched the previously known HLA-E genotype in 94 (44.3%) cell lines, in all cases either improving or equalling previous genotyping resolution. Three (1.4%) cells had discrepant HLA-E genotyping data and 115 (54.2%) had no previous HLA-E data. The HLA-E genotypes for four (1.9%) cell lines resulted in a change of zygosity by identifying two distinct haplotypes. We discovered eight novel HLA-E alleles, extended the known reference sequence of seven and confirmed the existence of a further 10.

Presence of donor-encoded centromeric KIR B content increases the risk of infectious mortality in recipients of myeloablative, T-cell deplete, HLA-matched HCT to treat AML.

The reported influence of donor Killer-cell Immunoglobulin-like Receptor (KIR) genes on the outcomes of haematopoietic cell transplantation (HCT) are contradictory, in part due to diversity of disease, donor sources, era and conditioning regimens within and between different studies. Here, we describe the results of a retrospective clinical analysis establishing the effect of donor KIR motifs on the outcomes of 119 HLA-matched, unrelated donor HCT for adult acute myeloid leukaemia (AML) using myeloablative conditioning (MAC) in a predominantly T-cell deplete (TCD) cohort. We observed that HCT involving donors with at least one KIR B haplotype were more likely to result in non-relapse mortality (NRM) than HCT involving donors with two KIR A haplotypes (p = 0.019). Upon separation of KIR haplotypes into their centromeric (Cen) and telomeric (Tel) motif structures, we demonstrated that the Cen-B motif was largely responsible for this effect (p = 0.001). When the cause of NRM was investigated further, infection was the dominant cause of death (p = 0.006). No evidence correlating donor KIR B haplotype with relapse risk was observed. The results from this analysis confirm previous findings in the unrelated, TCD, MAC transplant setting and imply a protective role for donor-encoded Cen-A motifs against infection in allogeneic HCT recipients.

Extending the sequences of HLA class I alleles without full-length genomic coverage using single molecule real-time DNA sequencing.

The assignment of an HLA allele name to a sequence requires a comparison between the generated target sequence and a reference sequence on the IPD-IMGT/HLA database. Absence of a full-length reference sequence can result in the inability of HLA typing software to accurately compare and assign the sequence. We sequenced the most frequently seen HLA class I alleles on the Anthony Nolan register present in the database with only a partial genomic sequence, with the aim of increasing the number of complete reference sequences. We successfully extended 95 full-length HLA class I sequences and identified 13 novel variants. Increasing the number of full-length HLA class I reference sequences in the database has aided accuracy of HLA analysis tools for all histocompatibility and immunogenetics laboratories.

A genomic extension to the sequence of HLA-A*02:13, identified using third-generation sequencing.

Pacific Biosciences' SMRT sequencing method was used to extend the sequence of HLA-A*02:13.

Identification of a new allele, HLA-DRB1*01:01:01:02 discovered using third-generation sequencing.

HLA-DRB1*01:01:01:02 is the first reported intronic variant of DRB1*01:01:01, differing by a single nucleotide substitution.

A novel allele, HLA-C*07:01:01:30 identified using third-generation sequencing.

The novel allele, HLA-C*07:01:01:30 differs by one nucleotide substitution with HLA-C*07:01:01:01 at gDNA 627.

Recipients Receiving Better HLA-Matched Hematopoietic Cell Transplantation Grafts, Uncovered by a Novel HLA Typing Method, Have Superior Survival: A Retrospective Study.

HLA matching at an allelic-level resolution for volunteer unrelated donor (VUD) hematopoietic cell transplantation (HCT) results in improved survival and fewer post-transplant complications. Limitations in typing technologies used for the hyperpolymorphic HLA genes have meant that variations outside of the antigen recognition domain (ARD) have not been previously characterized in HCT. Our aim was to explore the extent of diversity outside of the ARD and determine the impact of this diversity on transplant outcome. Eight hundred ninety-one VUD-HCT donors and their recipients transplanted for a hematologic malignancy in the United Kingdom were retrospectively HLA typed at an ultra-high resolution (UHR) for HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 using next-generation sequencing technology. Matching was determined at full gene level for HLA class I and at a coding DNA sequence level for HLA class II genes. The HLA matching status changed in 29.1% of pairs after UHR HLA typing. The 12/12 UHR HLA matched patients had significantly improved 5-year overall survival when compared with those believed to be 12/12 HLA matches based on their original HLA typing but were found to be mismatched after UHR HLA typing (54.8% versus 30.1%, P = .022). Survival was also significantly better in 12/12 UHR HLA-matched patients when compared with those with any degree of mismatch at this level of resolution (55.1% versus 40.1%, P = .005). This study shows that better HLA matching, found when typing is done at UHR that includes exons outside of the ARD, introns, and untranslated regions, can significantly improve outcomes for recipients of a VUD-HCT for a hematologic malignancy and should be prospectively performed at donor selection.

Next-generation HLA typing of 382 International Histocompatibility Working Group reference B-lymphoblastoid cell lines: Report from the 17th International HLA and Immunogenetics Workshop.

Extended molecular characterization of HLA genes in the IHWG reference B-lymphoblastoid cell lines (B-LCLs) was one of the major goals for the 17th International HLA and Immunogenetics Workshop (IHIW). Although reference B-LCLs have been examined extensively in previous workshops complete high-resolution typing was not completed for all the classical class I and class II HLA genes. To address this, we conducted a single-blind study where select panels of B-LCL genomic DNA samples were distributed to multiple laboratories for HLA genotyping by next-generation sequencing methods. Identical cell panels comprised of 24 and 346 samples were distributed and typed by at least four laboratories in order to derive accurate consensus HLA genotypes. Overall concordance rates calculated at both 2- and 4-field allele-level resolutions ranged from 90.4% to 100%. Concordance for the class I genes ranged from 91.7 to 100%, whereas concordance for class II genes was variable; the lowest observed at HLA-DRB3 (84.2%). At the maximum allele-resolution 78 B-LCLs were defined as homozygous for all 11 loci. We identified 11 novel exon polymorphisms in the entire cell panel. A comparison of the B-LCLs NGS HLA genotypes with the HLA genotypes catalogued in the IPD-IMGT/HLA Database Cell Repository, revealed an overall allele match at 68.4%. Typing discrepancies between the two datasets were mostly due to the lower-resolution historical typing methods resulting in incomplete HLA genotypes for some samples listed in the IPD-IMGT/HLA Database Cell Repository. Our approach of multiple-laboratory NGS HLA typing of the B-LCLs has provided accurate genotyping data. The data generated by the tremendous collaborative efforts of the 17th IHIW participants is useful for updating the current cell and sequence databases and will be a valuable resource for future studies.

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.