# Pitfalls When Determining HNA-1 Genotypes and Finding Novel Alleles

## TL;DR

This paper shows that current methods for determining HNA-1 genotypes are inaccurate, while long-read nanopore sequencing offers a more precise and comprehensive solution.

## Contribution

The study introduces long-read nanopore sequencing as a novel and specific method for FCGR3B genotyping, overcoming limitations of existing techniques.

## Key findings

- Long-read nanopore sequencing accurately detects FCGR3B alleles and dosage-dependent distributions.
- Current genotyping methods like TaqMan PCR and PCR-SSP produce ambiguous results due to co-amplification with FCGR3A.
- Nanopore sequencing can identify novel and rare FCGR3B variants previously undetected.

## Abstract

Genetic variation in the FCGR3B gene is responsible for different variants of human neutrophil antigen 1 (HNA-1). Laboratory techniques currently utilized for routine HNA-1 genotyping, predominantly PCR-sequence-specific primer (PCR-SSP) and PCR-sequence-based typing (PCR-SBT), lack specificity for FCGR3B. This study compares the capabilities and limitations of existing technologies including an in-house TaqMan PCR, a commercial PCR-SSP test, PCR-SBT and multiplex ligation-dependent probe amplification (MLPA) with those of a long-read nanopore sequencing assay. Testing was performed with both related and unrelated Danish samples with different copy numbers and/or rare alleles. Long-read nanopore sequencing was validated by blind testing of ten English samples. The results showed that FCGR3B copy numbers correlate with a dose-dependent distribution of alleles that complicates genotyping by TaqMan PCR, PCR-SSP and PCR-SBT, due to co-amplification of the homologous FCGR3A gene. MLPA can correctly quantify the dose-dependent distribution but not detect novel variants. Long-read nanopore sequencing showed high specificity for FCGR3B and was able to detect dosage-dependent distribution, and rare and novel variants that were previously not described. Current HNA-1 genotyping methods cannot produce unambiguous allele-level results, whereas long-read nanopore sequencing has shown the potential to resolve observed ambiguities, identify new HNA-1 variants and allow definitive allele assignment.