An accurate genetic assay to identify human neutrophil antigen 2 deficiency

Abstract

Objective: We aimed to develop accurate and user-friendly genetic assays to identify the inherited neutrophil antigen-2 (HNA-2) deficiency in humans.

Background: HNA-2 is one of the most important neutrophil antigens implicated in a number of human disorders. HNA-2 deficiency or HNA-2 null is a common phenotype observed in 3%-5% Americans. 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. We previously demonstrated that the CD177 coding SNP 787A > T (c.787A > T) is the most important genetic determinant for HNA-2 deficiency. However, reliable genetic assays are not available for routine clinical laboratory application up to now.

Study design and methods: 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.

Results: 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.

Conclusion: 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.

Keywords: CD177; HNA-2 deficiency; genetic assay; polymorphisms.

Fig. 1.. CD177 SNPs responsible for HNA-2 expression deficiency.

CD177 gene contains nine exons and the CD177 exon 4, 5, 6, 7, 8, and 9 are highly homologous to those of CD177 pseudogene (upper panel). Locations of CD177 SNPs responsible for HNA-2 expression deficiency are marked as vertical bars on cDNA in the middle panel. CD177 ORF/STP haplotypes are formed by five cSNPs (c.782G>C, c.786A>C, c.787A>T, c.790G>A, and c.799A>G) (lower panel).

Fig. 2.. PCR-based assay to determine ORF/STP genotypes.

a). Primer locations of PCR-based assay. A sense CD177 gene-specific primer (Sense GSP) was paired with an antisense CD177 ORF allele-specific primer and a sense CD177 STP allele-specific primer was paired with an antisense CD177 gene-specific primer (Antisense GSP) to yield allele-specific DNA fragments of 893 bps and 1,254 bps, respectively. b). The PCR products were separated on an agarose gel. The DNA fragment size of internal control (human growth hormone gene) is 429 bps. The CD177 genotypes were determined by the sizes and species of the DNA fragments in a single reaction. ORF-allele produces a DNA fragment of 893 bps. STP-allele produces a DNA fragment of 1,254 bps. c). CD177 ORF/STP genotypes were determined with the PCR-based genotyping assay on a cohort of human subject whose HNA-2 expression was examined with flow cytometry analysis using HNA-2 positive plasma. All STP homozygous donors (N = 6) were HNA-2 null. The percentages of HNA-2 positive neutrophils from ORF/STP heterozygous donors (N = 32) were significantly (P < 0.0001) lower than those from ORF homozygous donors (N = 68).

Fig. 3.. High-throughput CD177 ORF/STP TaqMan genotyping assay.

a). Primer locations of TaqMan assays to determine the genotypes of the CD177 SNP ORF or STP haplotypes. The CD177 gene-specific PCR products (2110 bps) were amplified with gene-specific primers (GSP) and subsequently used as TaqMan assay template. b). Plot of CD177 TaqMan genotyping assay. CD177 genotypes of the STP/ORF variant are clustered in three populations. The STP homozygous genotype is on the upper left corner, the ORF/STP heterozygous genotype in the middle, and the ORF/ORF homozygous on the lower right corner.