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

Area of Science:

• Immunogenetics
• Molecular Diagnostics

Background:

• Genetic variations in the FCGR3B gene determine human neutrophil antigen 1 (HNA-1) types.
• Current HNA-1 genotyping methods like PCR-SSP and PCR-SBT exhibit limited specificity for FCGR3B due to homology with FCGR3A.

Purpose of the Study:

• To compare the diagnostic capabilities of existing HNA-1 genotyping techniques with long-read nanopore sequencing.
• To evaluate the potential of nanopore sequencing for resolving ambiguities and identifying novel HNA-1 variants.

Main Methods:

• Comparative analysis of TaqMan PCR, PCR-SSP, PCR-SBT, and MLPA against long-read nanopore sequencing.
• Testing utilized Danish samples with varying FCGR3B copy numbers and rare alleles, validated with blind testing of English samples.

Main Results:

• Existing methods (TaqMan PCR, PCR-SSP, PCR-SBT) struggle with FCGR3B genotyping due to FCGR3A co-amplification, complicating allele distribution analysis.
• MLPA accurately quantifies FCGR3B copy number but fails to detect novel variants.
• Long-read nanopore sequencing demonstrated high specificity for FCGR3B, successfully identifying dosage variations, rare alleles, and previously undescribed variants.

Conclusions:

• Current HNA-1 genotyping methods are insufficient for unambiguous allele-level results.
• Long-read nanopore sequencing offers a promising solution for accurate HNA-1 genotyping, variant discovery, and definitive allele assignment.