Assessment of the relative number of copies of the gene encoding human neutrophil antigen-2a (HNA-2a), CD177, and a homologous pseudogene by quantitative real-time PCR

Abstract

Abstract Human neutrophil antigen-2a (HNA-2a; NB1) is located on the 58–64 kD NB1 glycoprotein (GP) and is encoded by the gene CD177. Searches of human genome databases have revealed that a pseudogene highly homologous to exons 4–9 of CD177 is located adjacent to CD177 on chromosome 19. The purpose of this study was to document the presence of the pseudogene and determine whether the polymorphic expression of NB1 GP is due to CD177 gene deletions and duplications. Genomic DNA was isolated from leukocytes of 12 subjects. The number of copies of exon 2 of CD177, an exon that is unique to this gene, and the number of copies of exon 9, an exon that is found in both CD177 and the pseudogene, was assessed with quantitative real-time PCR. The ratio of the number of copies of sequences homologous to CD177 exon 9 to the number of copies of exon 2 was 1.5 or greater in 7 of the 12 subjects, suggesting that both CD177 and the homologous pseudogene were present. The ratio of exon 9 to exon 2 in the other 5 subjects ranged from 1 to 1.25, suggesting that the pseudogene was not present in these subjects. However, results of assays were variable and we could not exclude the possibility that all subjects carried the pseudogene. These studies confirmed the presence of the pseudogene homologous to CD177, but quantitative real-time PCR was not precise enough to detect CD177 duplications or deletions.