Accurate Genetic Assay Identifies Human Neutrophil Antigen 2 Deficiency

HNA-2 null individuals are at risk to produce isoantibodies (or alloantibodies) that play important roles in transfusion-related acute lung injury, immune neutropenia, and bone marrow graft failure. CD177 coding SNP 787A > T (c.787A > T) is the most important genetic determinant for HNA-2 deficiency that involves human myeloproliferative disorders.

Biomedical Scientists at the University of Minnesota (St. Paul, MN, USA) recruited healthy blood donors at the Memorial Blood Center in St. Paul, MN, USA). The age of healthy control donors ranged from 19 to 84 years old. Human genomic DNA was isolated from EDTA anti-coagulated peripheral blood using the Wizard Genomic DNA Purification kit (Promega, Madison, WI, USA).

The expression of HNA-2 and the percentage of HNA-2+ neutrophils in healthy blood donors were determined. Fresh whole blood samples were stained with FITC-conjugated mouse anti-human CD177 (HNA-2) mAb MEM-166 or FITC-conjugated mIgG1 isotype control and analyzed on a FACS Canto flow cytometer (BD Biosciences, San Jose, CA, USA). A novel polymerase chain reaction (PCR) strategy was used to determine genotypes of the CD177 SNP c.787A > T.

In the simplified PCR assay, all allele specific primers and internal control primers were included in the same reaction, which ensures reliability of the assay. In addition, a novel high-throughput nested TaqMan assay was developed to determine genotypes of c.787A > T for large population genetic analysis of HNA-2 deficiency. The Applied Biosystems Veriti 96-well Thermal Cycler was used for the PCR reactions (Thermo Fisher Scientific, Waltham, MA, USA).

The scientists reported that CD177 SNP c787A > T genotypes of 396 subjects were 100% concordant among the single PCR reaction method, the nested TaqMan assay, and Sanger Sequencing analysis. Out of 396 subjects, all 18 donors with the CD177 STP homozygous genotype were HNA-2 null.

The authors concluded that the novel PCR-based genotyping assay is accurate to identify HNA-2 deficient individuals and is suitable for clinical laboratories. In addition, the innovative high-throughput nested TaqMan assay will be useful for large-scale population screens and genetic studies of HNA-2 deficiency. The study was on October 29, 2022 in the journal Transfusion Medicine.