Structure and Function of Granulocyte Antigens

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Abstract

Granulocyte antigens play an important role in cell functions including adhesion, cell activation, and binding of immunoglobulins. The purpose of these studies is to better define the molecular basis of variations in neutrophil antigens and their role in neutrophil function. Neutrophil-specific antigen HNA-2a (NB1) has been localized to CD177 glycoprotein (gp) which is expressed on subpopulations of neutrophils. PRV-1 is a gene that is over expressed in neutrophils from patients with polycythemia rubra vera. The gene encoding NB1 differs from PRV-1 at four reported nucleotides. The goal of this study was to determine if PRV-1 and NB1 were alleles of the same gene or two separate genes; moreover, if they are alleles of the same gene, to determine the gene frequencies of each allele and explore potential correlations to neutrophil CD177 gp expression. Primer pairs were used to amplify leukocyte genomic DNA in the regions surrounding the four NB1 polymorphic sites within exon 1, exon 3, exon 8, and exon 9. The four resulting amplicons were sequenced and analyzed for each donor. The size of the neutrophil population in each donor staining brightly with CD177 antibody was assessed by flow cytometry. If PRV-1 and NB1 are separate genes, then all people tested should be heterozygous for the PRV-1/NB1 polymorphisms. Since 6 of 16 donors tested were homozygous for PRV-1 polymorphisms at all 4 sites, PRV-1 and NB1 are alleles of the same gene, CD177. When the sequenced exons in the 16 donors were compared to PRV-1, 14 single nucleotid polymorphisms (SNPs) were found. Thirteen of the 14 SNPs result in amino acid changes. The G42C exon 1 NB1 polymorphisms was the most common SNP. It was found in 7 donors. The 7 SNPs in exons 1, 2 and 3 are in the CD177 gene, but it is uncertain if the 7 SNPs in exons 7, 8, and 9 are in CD177 or a homologous pseudo gene. Thirty-two SNPs were also found in introns 2, 7, and 8. Since the G42C SNP resulted in an amino acid change in the CD177 protein leader sequence the size of the CD177 bright neutrophil population was compared among donors homozygous of G at bp42, 42GG, and those homozygous for C, 42CC. The CD177 bright neutrophil population was greater in 42CC donors than in 42GG donors. These studies show that PRV-1 and NB1 are alleles of the polymorphic gene CD177. The most common SNP in bp42 predicted an amino acid change in the protein's leader sequence and effects protein expression. Future studies will be directed toward evaluating the entire CD177 gene sequence in normal donors and in patient with polycythemia vera.