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

Authors :
Kløve-Mogensen, Kirstine
Browne, Tom
Haunstrup, Thure Mors
Steffensen, Rudi
Source :
International Journal of Molecular Sciences. Aug2024, Vol. 25 Issue 16, p9127. 18p.
Publication Year :
2024

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. [ABSTRACT FROM AUTHOR]

Details

Language :
English
ISSN :
16616596
Volume :
25
Issue :
16
Database :
Academic Search Index
Journal :
International Journal of Molecular Sciences
Publication Type :
Academic Journal
Accession number :
179349306
Full Text :
https://doi.org/10.3390/ijms25169127