Pitfalls When Determining HNA-1 Genotypes and Finding Novel Alleles

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.

Keywords: FCGR3A; FCGR3B; HNA-1; PCR; copy number variation; genotyping; long-read sequencing; nanopore.

Figure 1

TaqMan cluster plot analysis for c.114C/T and c.316A/G, both showing indication of five clusters. Note: Highlighted blue and red circles are the homozygous clusters, and green circles are the heterozygous cluster representing a sample with two copies. In the spaces between are two intermediate clusters highlighted with purple and yellow representing clusters with heterozygosity but containing three nucleotides.

Figure 2

Segregation of a novel FCGR3B allele found in AAL_003 (I-2) to AAL_013 (II-3). Note: The first generation is marked with I and the second with II. Male individuals are marked by square symbols, and females by circles. The individuals’ copy numbers (CN) are shown inside the symbols. The five SNPs and their genotypes are shown for each allele. Alleles inherited together are positioned inside the same bracket.

Figure 3

Neutrophil phenotyping with human antisera for the blood donor with a novel HNA-1 (FCGR3B) allele (BRI_007). Note: Previously presented by Browne, T., 2022 [49]. (a) Isotype control and known negative antisera. (b) GI 10 in-house CD16b-specific antisera. (c) GI 21 in-house HNA-1a-specific antisera. (d) GI 2 in-house HNA-1b-specific antisera. All murine monoclonal antibodies and human antisera specific for CD16b were positive. All CD16b-1a and CD16b-1b human antisera and CD16b-1a monoclonal antibody were negative.

Figure 4

Study I: Segregation of the FCGR3B*Null allele within two generations. Note: The first generation is marked with I and the second with II. Male individuals are marked by square symbols, and females by circles. The individuals’ copy numbers (CN) are shown inside the symbols. The five SNPs and their genotypes are shown for each allele.

Figure 5

Study II: Segregation of the FCGR3B*01 and FCGR3B*02 together through two generations. Note: The first generation is marked with I and the second with II. Male individuals are marked by square symbols, and females by circles. The individuals’ copy numbers (CN) are shown inside the symbols. The five SNPs and their genotypes are shown for each allele. Alleles inherited together are positioned inside the same bracket.

Figure 6

Study III: Segregation of the FCGR3B*03 and FCGR3B*04 together through three generations. Note: The first generation is marked with I and the second with II. Male individuals are marked by square symbols, and females by circles. The individuals’ copy numbers (CN) are shown inside the symbols. The five SNPs and their genotypes are shown for each allele. Alleles inherited together are positioned inside the same bracket.

Figure 7

Study IV: Segregation of the FCGR3B*03 and FCGR3B*04 together. Note: The first generation is marked with I and the second with II. Male individuals are marked by square symbols, and females by circles. The individuals’ copy numbers (CN) are shown inside the symbols. The five SNPs and their genotypes are shown for each allele. Alleles inherited together are positioned inside the same bracket.