P030How do different testing platforms influence novel allele characterization? A case study of a novel DPB1 allele

A renal transplant patient sample was submitted in order to characterize an unresolved HLA-DPB1 allele. Sanger sequence-based typing (SBT) resulted in DPB1*04:02:01 and a number of equally possible candidates for the second allele, each with a single nucleotide mismatch. Removing all exons except exon 2 from analysis resulted in a perfect match with homozygous DPB1*04:02:01G. However, several heterozygous positions leading to non-synonymous mutations were present in the excluded exons, contradicting this apparent homozygosity. One next-generation sequencing (NGS) platform then typed the sample as DPB1*04:02:01 for the first allele and various ambiguous alleles for the second. However, this was due to low coverage in a 19-nucleotide region of exon 2 rather than a mismatch. The low coverage pattern was replicated on a repeat run. DPB1*414:01 with one mismatch in exon 2 was called as the second allele when the minimum read depth was lowered. Interestingly, none of the possible second alleles were part of DPB1*04:02:01G. Upon testing on a second NGS platform, DPB1*04:02:01, DPB1*463:01:01 was obtained with novel mutations in exon 4 and introns. Comparing this result to the Sanger SBT data revealed the same heterozygous mismatch in exon 4. A third NGS platform verified this typing and associated mismatches. Therefore, if potential intronic mutations are ignored, the typing may be reported in two ways: as DPB1*04:02:01, DPB1*463:01 with a mutation in exon 4, or as DPB1*04:02:01, DPB1*414:01 with a mutation in exon 2. The first combination may lead to the assumption of homozygosity for treatment purposes, since DPB1*463:01 is part of DPB1*04:02:01G. This is currently the practice in the HLA field when exons 3 and 4 are not included in analysis. Ignoring heterozygous positions in exons that make up regions outside the antigen binding groove could lead to development of antibodies. This may be an important consideration in the renal transplant setting, depending on patient-donor matching and determination of immunogenicity.