Identification of the novel HLA-DRB1*15:184 allele by next-generation sequencing.

HLA-DRB1*15:184 differs from HLA-DRB1*15:01:01 by a single base substitution in exon 3 at codon 134.

Human neutrophil antigen 2 sequence-based typing: Joining the hunt for the CD177 answer.

BACKGROUND AND OBJECTIVES: Isoantibodies to human neutrophil antigen 2 (CD177) have been associated with several clinical conditions but to date the molecular basis for altered or non-expression has not been determined. Reliance on phenotyping and crossmatch to investigate these neutropenic clinical cases are inconvenient for the patients and demanding of resources within the laboratory. Therefore, a molecular approach has been introduced to address both issues. MATERIALS AND METHODS: A DNA panel of 100 randomly selected blood donors were collected and supplemented with 18 DNA samples from blood donors previously shown to be CD177 null. All DNA samples were sequence-based typed for all exons and observed polymorphisms recorded. The DNA from two families previously investigated for neonatal alloimmune neutropenia due to CD177 isoantibodies were also analysed. RESULTS: The incidence of CD177 null could be associated with a known exon 7 single-nucleotide polymorphism in 16/21 known CD177 null samples, which is consistent with previously published findings. Two additional mutations that may lead to null expression were also identified, of which one may be novel. In both family investigations, this same mutation could also be observed in the maternal DNA sample. CONCLUSION: Based on these observations, introduction of CD177 genotyping into routine use would identify null expression in over 75% (16/21) of associated cases. In turn, this could significantly reduce the need for supplementary testing and associated inconvenience to patients while permitting increased efficiency of laboratory testing. An added benefit would potentially elucidate other clinically relevant mutations and associated antigenic targets.

Identification of the novel HLA-B*42:28 allele by next-generation sequencing.

HLA-B*42:28 differs from HLA-B*42:01:01 by a single base substitution in exon 4 at codon 267.2.

The first reported case of neonatal alloimmune thrombocytopenia due to low-frequency human platelet antigen-6b antibodies in the United Kingdom.

BACKGROUND: Neonatal alloimmune thrombocytopenia (NAIT) is a potentially serious clinical condition caused by maternal alloantibodies directed to human platelet antigens (HPA), inherited from the father and expressed on fetal/neonatal platelets. We report a case of an otherwise well, full term child, with a profound thrombocytopenia (33 x 109/L). There was no bleeding or obvious explanation for the low platelet count. Samples were sent for the investigation of NAIT. METHOD: Serological investigations were performed on maternal serum taken at day (D)+4 and D+78. The platelet immunofluorescence test (PIFT) and monoclonal antibody immobilization of platelet antigens (MAIPA) assays were performed with a panel of HPA typed donor platelets and against paternal platelets in a crossmatch. HPA 1-6, -9 and -15 and HLA genotyping was performed by in-house PCR-sequence based typing (SBT) and next generation sequencing (NGS). RESULTS: HPA antibody screening of D+4 maternal serum indicated that platelet-specific antibodies were absent. HPA genotyping of the father and child revealed the presence of the low frequency HPA antigen (LFHPA), HPA-6b, which was absent in the mother. Maternal samples were crossmatched against paternal platelets and were positive by PIFT and glycoprotein (GP) IIb/IIIa and HLA class I in the MAIPA assay. The infant required no platelet transfusion support as the thrombocytopenia resolved spontaneously. DISCUSSION: We conclude that the positive crossmatch reaction was due to anti-HPA-6b alloantibodies. This case further emphasizes the importance of platelet crossmatching and HPA genotyping of LFHPA in cases where there is a high clinical suspicion of NAIT but initial screening is negative.

Rapid, highly accurate and cost-effective open-source simultaneous complete HLA typing and phasing of class I and II alleles using nanopore sequencing.

Accurate rapid genotyping of the genes within the HLA region presents many difficulties because of the complexity of this region. Here we present the results of our proof of concept nanopore-based long read polymerase chain reaction (PCR) solution for HLA genotyping. For 15 HLA anthropology-based samples and 13 NHS Blood and Transplant derived samples 40 ng of genomic DNA underwent long-range PCR for class I and II HLA alleles. Pooled PCR products were sequenced on the Oxford Nanopore MinIoON R9.4.1 flow cell. Sequenced reads had HLA genotype assigned with HLA-LA. Called genotypes were compared with reference derived from a combination of short-read next-generation sequencing, Sanger sequence and/or single-site polymorphism (SSP) typing. For concordance, accuracy was 100%, 98.4%, 97.5% and 95.1% for the first, second, third and fourth fields, respectively, to four field accuracy where it was available, otherwise three field in 28 samples for class I calls and 17 samples for class II calls. Phasing of maternal and paternal alleles, as well as phasing based identification of runs of homozygosity, was shown successfully. Time for assay run was 8 hours and the reconstruction of HLA typing data was 15 minutes. Assay cost was £55 ($80USD)/sample. We have developed a rapid and cost-effective long-range PCR and nanopore sequencing-based assay that can genotype the genes within HLA region to up to four field accuracy, identify runs of homozygosity in HLA, reconstruct maternal and paternal haplotypes and can be scaled from multi-sample runs to a single sample.