Aim: During a confirmatory NGS-HLA typing of an NMDP donor, we discovered a discrepancy at DQB1*. The registry typing for the DQB1* locus was heterozygous demonstrating DQB1*02:02 and DQB1*06:02. Our typing of the sample, utilizing the GenDx MX11-3 HLA-specific typing kit on the Illumina MiSeq system, showed an apparent DQB1*06:02 homozygous with no indication of a second allele.
Method: A qPCR on the sample was run to determine if the predicted second allele was present. Analysis of the qPCR results did show the presence of the second allele (DQB1*02). Our NGS data files was sent to GenDx support to confirm our analysis. GenDx support found that the sample had high read depth, low noise levels and a good separation between signal to noise and confirmed our results of DQB1*06:02 homozygous with the suggestion to repeat the sample from amplification. We then performed an additional NGS typing with GenDx NGS Turbo utilizing the MinION platform.
Results: The additional NGS typing with GenDx NGS Turbo did show DQB1*02:02 and DQB1*06:02. An additional repeat sample using MX11-3 again did not show any signs of a second allele at DQB1. A suspected single nucleotide polymorphism (SNP) under the primer binding site for the MX11-3 primers for the amplification of the DQB1 allele was thought to be present. This SNP will lead to the inability to amplify the DQB1 allele and eventually the observed allele drop-out.
Conclusion: This case demonstrated that some HLA alleles may suffer dropouts due to polymorphisms in the binding region of the primers for long-range amplification. This demonstrates the need for labs to remain vigilant for appropriate linkages and haplotypes, as well as the need for additional platforms to confirm certain apparent homozygosities.
