Determination of CD177 (human neutrophil antigen 2) polymorphisms using nanopore sequencing.

BACKGROUND AND OBJECTIVES

Human neutrophil antigen 2 (HNA-2), encoded by the CD177 gene, is considered one of the most important neutrophil antigens in human medicine, but molecular testing of CD177 is complicated and therefore not a standard procedure for investigating CD177 expression. CD177 expression can vary from 0% to 100%, and to date, the molecular basis for altered or non-expressed genes has not been determined. Reliance on phenotyping and crossmatching to investigate these neutropenic clinical cases is inconvenient for patients and demands substantial resources within the laboratory. The purpose of this study was therefore to test a new molecular testing approach based on long-read nanopore sequencing.

MATERIALS AND METHODS

DNA from 44 Danish blood donors with different levels of CD177 expression, 22 of whom were found to be CD177 null, was selected as test samples. All the DNA was sequenced for the first eight exons and the beginning of exon 9 of CD177.

RESULTS

All incidences of CD177 null cases could be associated with the known variant c.787A>T;p.K263X (rs20182172), and a correlation was observed between c.787A>T heterozygosity and a reduced expression of CD177, which is consistent with previously published findings. The c.1291G>A;p.G431R (rs78718189) variant was found to be linked to the atypical expression of CD177. The nanopore assay revealed a total of 14 variants in 7 exons in the 44 tested samples.

CONCLUSION

On the basis of these observations, we conclude that long-read nanopore sequencing can be a reliable tool for the routine laboratory molecular testing of CD177.