Novel alleles in the era of next-generation sequencing-based HLA typing calls for standardization and policy.

Next-Generation Sequencing (NGS) has transformed clinical histocompatibility laboratories through its capacity to provide accurate, high-throughput, high-resolution typing of Human Leukocyte Antigen (HLA) genes, which is critical for transplant safety and success. As this technology becomes widely used for clinical genotyping, histocompatibility laboratories now have an increased capability to identify novel HLA alleles that previously would not be detected using traditional genotyping methods. Standard guidelines for the clinical verification and reporting of novelties in the era of NGS are greatly needed. Here, we describe the experience of a clinical histocompatibility laboratory's use of NGS for HLA genotyping and its management of novel alleles detected in an ethnically-diverse population of British Columbia, Canada. Over a period of 18 months, 3,450 clinical samples collected for the purpose of solid organ or hematopoietic stem cell transplantation were sequenced using NGS. Overall, 29 unique novel alleles were identified at a rate of ∼1.6 per month. The majority of novelties (52%) were detected in the alpha chains of class II (HLA-DQA1 and -DPA1). Novelties were found in all 11 HLA classical genes except for HLA-DRB3, -DRB4, and -DQB1. All novelties were single nucleotide polymorphisms, where more than half led to an amino acid change, and one resulted in a premature stop codon. Missense mutations were evaluated for changes in their amino acid properties to assess the potential effect on the novel HLA protein. All novelties identified were confirmed independently at another accredited HLA laboratory using a different NGS assay and platform to ensure validity in the reporting of novelties. The novel alleles were submitted to the Immuno Polymorphism Database-Immunogenetics/HLA (IPD-IMGT/HLA) for official allele name designation and inclusion in future database releases. A nationwide survey involving all Canadian HLA laboratories confirmed the common occurrence of novel allele detection but identified a wide variability in the assessment and reporting of novelties. In summary, a considerable proportion of novel alleles were identified in routine clinical testing. We propose a framework for the standardization of policies on the clinical management of novel alleles and inclusion in proficiency testing programs in the era of NGS-based HLA genotyping.

High-throughput sequencing defines donor and recipient HLA B-cell epitope frequencies for prospective matching in transplantation.

Compatibility for human leukocyte antigen (HLA) genes between transplant donors and recipients improves graft survival but prospective matching is rarely performed due to the vast heterogeneity of this gene complex. To reduce complexity, we have combined next-generation sequencing and in silico mapping to determine transplant population frequencies and matching probabilities of 150 antibody-binding eplets across all 11 classical HLA genes in 2000 ethnically heterogeneous renal patients and donors. We show that eplets are more common and uniformly distributed between donors and recipients than the respective HLA isoforms. Simulations of targeted eplet matching shows that a high degree of overall compatibility, and perfect identity at the clinically important HLA class II loci, can be obtained within a patient waiting list of approximately 250 subjects. Internal epitope-based allocation is thus feasible for most major renal transplant programs, while regional or national sharing may be required for other solid organs.