Concurrent typing of over 4000 samples by long-range PCR amplicon-based NGS and rSSO revealed the need to verify NGS typing for HLA allelic dropouts

• Published in: Human immunology 2021-08, Vol.82 (8), p.581-587
• Main Authors: Kong, Denice, Lee, Nancy, Dela Cruz, Imma Donna, Dames, Charlyn, Maruthamuthu, Stalinraja, Golden, Todd, Rajalingam, Raja
• Format: Article
• Language: English
• Subjects: Alleles; Allelic dropout; Haplotypes; Hematopoietic Stem Cell Transplantation; High-resolution HLA typing; High-Throughput Nucleotide Sequencing - methods; Histocompatibility; Histocompatibility Testing - methods; Histocompatibility Testing - standards; HLA Antigens - genetics; HLA matching; Humans; Next-generation sequencing of HLA; Oligonucleotides; Polymerase Chain Reaction - methods; Polymerase Chain Reaction - standards
• ISSN: 0198-8859; 1879-1166
• DOI: 10.1016/j.humimm.2021.04.008

Hematopoietic stem cell transplantation (HSCT) from HLA-matched donors significantly decreases the risks of graft-rejection and graft-versus-host disease. Long-range PCR- amplicon-based next-generation sequencing (NGS) is increasingly used as a standalone method in clinical laboratories to determine HLA compatibility for HSCT and solid-organ transplantation. We hypothesized that an allelic dropout is a frequent event in the long-range PCR amplicon-based NGS HLA typing method. To test the hypothesis, we typed 4,006 samples concurrently using a commercially available long-range PCR amplicon-based NGS-typing and short exon-specific amplicon-based reverse sequence-specific oligonucleotide (rSSO) methods. The concordance between the NGS and rSSO typing results was 100% at HLA-A, -B, -C, -DRB1, -DRB3, -DRB5, -DQA1, DPA1 loci. However, 4.5% of the samples (179/4006) showed allelic-dropouts at one of the other three loci: HLA-DRB4 (3.9%), HLA-DPB1 (0.4%), and HLA-DQB1*(0.15%). The allelic-dropouts are not associated with specific haplotypes, and some dropouts can be reagent lot-specific. Although DRB1-DRB3/4/5-DQB1 linkages help to diagnose these allelic-dropouts in some cases, the rSSO typing was crucial to identify the dropouts in DQB1 and DPB1 loci. These results uncover the critical limitations of using long-range PCR amplicon-based NGS as a standalone method in clinical histocompatibility laboratories and advocate the need for strategies to diagnose and resolve allelic-dropouts.