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 NB1 glycoprotein (gp) which is expressed on subpopulations of neutrophils and is encoded by the CD177 gene. The gene encoding the NB1 gp was recently sequenced and called NB1. Another group described a gene which they called PRV-1 that is highly homologous to NB1. PRV-1 is a gene that is over expressed in neutrophils from patients with polycythemia rubra vera. The gene encoding PRV-1 differs from NB1 at four nucleotides. Previous work from our laboratory found that searches of human genome databases suggested that NB1 and PRV-1 were the same gene located on chromosome 19q13.2. In addition, database searches revealed that a pseudo gene homologous to exons 4 through 9 of PRV-1 was located on chromosome 19q13.2.
The aim of these laboratory investigations was to determine if PRV-1 and NB1 are alleles of the same gene and if other genes homologous to PRV-1 are present in the human genome. The coding region of PRV-1 was amplified from human fetal liver total RNA by PCR, cloned into a TOPO plasmid was used as a probe to screen a human bacterial artificial chromosme (BAC) library prepared with somatic DNA from a single individual (RPCI-11). Five BACs in the RPCI-11 library were reactive with the PRV-1 probes. The BACs identified in the library were analyzed by PCR and sequencing. Sequencing of the 5? and 3? termini of the BACs found that all 5 were located on chromosome 19 and 4 of the 5 had the same 5? and 3? termini sequences. PCR analysis of all 5 BACs rendered an amplicon when amplified with a primer pair encompassing PRV-1 exons 1 and 2 and a second primer pair encompassing exons 7 and 8. Sequencing revealed that both amplicons in all 5 BACs contained sequences homologous to PRV-1. In all 5 BACs the fragment containing exons 1 and 2 provided the same unique sequence that was identical to PRV-1. The fragment encompassing exons 7 and 8 proved to be two fragments, with similar but heterozygous sequences, consistent with the presence of a pseudogene. One BAC containing PRV-1 was used in a fluorescence in situ hybridization (FISH) assay to localize genes homologous to PRV-1 within the human genome. FISH analysis showed that genes homologous to PRV-1 are only located on chromosome 19q13,2. These results show that PRV-1 and NB1 are alleles of the same polymorphic gene, CD177, that is located on chromosome 19q13.2. The coding region of CD177 from 3 healthy subjects was cloned and sequenced. Four of the six halpotypes were identical to PRV-1. The other two haplotypes each differed from PRV-1 at a single nucleotide. In conclusion, PRV-1 is the most common allele of CD177. The increased levels of neutrophil CD177 mRNA found polycythemia vera are likely a marker of increased myelopoiesis in addition to a marker of myeloproliferation.
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