Gene and microRNA analysis of neutrophils from patients with polycythemia vera and essential thrombocytosis: down-regulation of micro RNA-1 and -133a.

BACKGROUND: Since the V617F mutation in JAK2 may not be the initiating event in myeloprofilerative disorders (MPDs) we compared molecular changes in neutrophils from patients with polycythemia vera (PV) and essential thrombocythosis (ET), to neutrophils stimulated by G-CSF administration and to normal unstimulated neutrophils METHODS: A gene expression oligonucleotide microarray with more than 35,000 probes and a microRNA (miR) expression array with 827 probes were used to assess neutrophils from 6 MPD patients; 4 with PV and 2 with ET, 5 healthy subjects and 6 healthy subjects given G-CSF. In addition, neutrophil antigen expression was analyzed by flow cytometry and 64 serum protein levels were analyzed by ELISA. RESULTS: Gene expression profiles of neutrophils from the MPD patients were similar but distinct from those of healthy subjects, either unstimulated or G-CSF-mobilized. The differentially expressed genes in MPD neutrophils were more likely to be in pathways involved with inflammation while those of G-CSF-mobilized neutrophils were more likely to belong to metabolic pathways. In MPD neutrophils the expression of CCR1 was increased and that of several NF-kappaB pathway genes were decreased. MicroRNA miR-133a and miR-1 in MPD neutrophils were down-regulated the most. Levels of 11 serum proteins were increased in MPD patients including MMP-10, MMP-13, VCAM, P-selectin, PDGF-BB and a CCR1 ligand, MIP-1alpha. CONCLUSION: These studies showed differential expression of genes particularly involved in inflammatory pathways including the NF-kappaB pathway and down-regulation of miR-133a and miR-1. These two microRNAs have been previous associated with certain cancers as well as the regulation of hyperthrophy of cardiac and skeletal muscle cells. These changes may contribute to the clinical manifestations of the MPDs.

Identification of HLA-A33-restricted CMV pp65 epitopes as common targets for CD8(+) CMV-specific cytotoxic T lymphocytes.

OBJECTIVE: To identify an immunodominant HLA-A33-restricted epitope within the CMV matrix phosphoprotein 65 (pp65) that could be used to elicit CMV-specific CTLs. METHODS: A computer algorithm was used to identify pp65 peptides that were expected to bind to HLA-A33. The peptides were screened for their ability to reactivate memory T lymphocytes from CMV-seropositive HLA-A33 donors by using quantitative real-time RT-PCR. The most promising peptides were then tested for their ability to expand a CD8(+) population of HLA-A33 CTLs that produced interferon-gamma (IFN-gamma) and were cytotoxic to either peptide-loaded or CMV-infected target cells. RESULTS: Sixteen out of 250 peptides were selected using a computer algorithm and peptide stimulation by 8 out of the 16 induced a significant quantity of IFN-gamma mRNA transcript from CMV-seropositive HLA-A33 peripheral blood mononuclear cells. All the eight peptides induced consistent T-cell reactivation. One specifically, the peptide pp65(91-100) (SVNVHNPTGR), proved to be more active. T cells in vitro sensitized for 2 or 3 weeks with pp65(91-100) were cytotoxic to both HLA-A33 peptide-loaded and CMV-infected target cells. CONCLUSIONS: CMV pp65(91-100) is a new HLA-A33-restricted peptide that broadens the list of antigenic determinants that can be used for CMV adoptive immunotherapy.

CD177: A member of the Ly-6 gene superfamily involved with neutrophil proliferation and polycythemia vera.

Genes in the Leukocyte Antigen 6 (Ly-6) superfamily encode glycosyl-phosphatidylinositol (GPI) anchored glycoproteins (gp) with conserved domains of 70 to 100 amino acids and 8 to 10 cysteine residues. Murine Ly-6 genes encode important lymphocyte and hematopoietic stem cell antigens. Recently, a new member of the human Ly-6 gene superfamily has been described, CD177. CD177 is polymorphic and has at least two alleles, PRV-1 and NB1. CD177 was first described as PRV-1, a gene that is overexpressed in neutrophils from approximately 95% of patients with polycythemia vera and from about half of patients with essential thrombocythemia. CD177 encodes NB1 gp, a 58-64 kD GPI gp that is expressed by neutrophils and neutrophil precursors. NB1 gp carries Human Neutrophil Antigen (HNA)-2a. Investigators working to identify the gene encoding NB1 gp called the CD177 allele they described NB1. NB1 gp is unusual in that neutrophils from some healthy people lack the NB1 gp completely and in most people NB1 gp is expressed by a subpopulation of neutrophils. The function of NB1 gp and the role of CD177 in the pathogenesis and clinical course of polycythemia vera and essential thrombocythemia are not yet known. However, measuring neutrophil CD177 mRNA levels has become an important marker for diagnosing the myeloproliferative disorders polycythemia vera and essential thrombocythemia.

Another example of a KEL1 variant red cell phenotype due to a threonine to serine change at position 193 of Kell glycoprotein.

BACKGROUND: Healthy subjects whose red blood cells (RBCs) react variably with anti-KEL1, but strongly express other Kell blood group antigens, have been described and called KEL1 variant. A 53-year-old Caucasian blood donor was identified whose RBCs reacted with three monoclonal and two polyclonal anti-KEL1 and did not react with two monoclonal and one polyclonal anti-KEL1. The molecular basis of this phenotype was investigated. STUDY DESIGN AND METHODS: Genomic white blood cell DNA was analyzed for KEL*1/2 genotype by utilizing sequence-specific primers and polymerase chain reaction. In addition, the region of the KEL*1/2 polymorphism at position 578 of KEL was analyzed by DNA sequencing. RESULTS: Genotyping of the donor with the KEL1 variant phenotype revealed that he was KEL*2 homozygous. Sequencing revealed one typical KEL*2 allele and a KEL*2 allele with an adenosine (A) to thymidine (T) substitution at position 577 that predicted a threonine to serine change at position 193. CONCLUSION: It is not known if this phenotype is clinically relevant, but for at least some genotyping applications probes that identify this polymorphism should be used and anti-KEL1 should be tested for reactivity to this allele.

The gene overexpressed in polycythemia rubra vera, PRV-1, and the gene encoding a neutrophil alloantigen, NB1, are alleles of a single gene, CD177, in chromosome band 19q13.31.

BACKGROUND: PRV-1 mRNA is overexpressed by neutrophils from polycythemia vera patients and is homologous to NB1 a gene overexpressed in reactive neutrophilia. STUDY DESIGN AND METHODS: These investigations were designed to confirm searches of genome databases suggesting that PRV-1 and NB1 are alleles of the same gene, CD177, and confirm a pseudogene adjacent to CD177. Methods included polymerase chain reaction (PCR), cloning, sequencing, and fluorescent hybridization studies. RESULTS: The coding region of PRV-1 was PCR-amplified from human fetal RNA, cloned, and used to screen the RPCI-11 bacterial artificial chromosome (BAC) library. Five BACs were reactive with the PRV-1 probe. PCR analysis of the BACs with primers encompassing PRV-1 exons, containing four known single-nucleotide polymorphisms, followed by sequencing rendered amplicons identical to PRV-1 in all five BACs. Analysis of all five by restriction digestion yielded fragments possible only if both the gene and the pseudogene are present. End sequencing of the BACs localized them to the same chromosome region. G-banding and fluorescence in situ hybridization at the 400- and 850-band levels of resolution mapped one BAC to chromosome band 19q13.2 and sublocalized the BAC to band 19q13.31, respectively. CONCLUSION: PRV-1 and NB1 are alleles of the same gene now referred to as CD177. Changes in CD177 expression may be a marker of increased or decreased myelopoiesis and are therefore an effect of, rather than a cause of, myeloproliferative disorders.

KEL6 and KEL7 genotyping with sequence-specific primers.

BACKGROUND: Although antibodies to Js(a) and Js(b) are clinically significant, reagent-quality anti-Js(a) and anti-Js(b) are not readily available. A sequence-specific primer-polymerase chain reaction (SSP-PCR) genotyping assay was tested that makes use of two single-nucleotide polymorphisms (SNPs) at positions 1910 and 2019 of KEL. These SNPs distinguish the gene encoding Js(a), KEL6; and Js(b), KEL7. STUDY DESIGN AND METHODS: Four primer sets that selectively amplified KEL6 and KEL7 from genomic DNA were developed. Two sets detected the SNP at bp 1910 and two sets detected the bp 2019 SNP. KEL6 and KEL7 genotyping and Js(a) and Js(b) phenotyping results were compared among 64 subjects. RESULTS: The SSP-PCRs were specific for KEL6 and KEL7 when testing DNA for three donors of known Js phenotype: Js(a+b-), Js(a-b+), and Js(a+b+). Genotyping results for the 1910 SNP were identical to the phenotyping results in all 64 subjects, but for the 2019 SNP, the genotyping and phenotyping results were identical for only 49 subjects. In 12 subjects with the Js(a-b+) phenotype, the 2019 SNP was heterozygous KEL6, KEL7; in 2 with Js(a-b+) and in 1 with Js(a+b+), the 2019 SNP was homozygous KEL6. CONCLUSION: KEL 2019-bp SNP does not always correlate with the Js phenotype owing to the presence of an atypical KEL gene with a KEL7 polymorphism at 1910 and a KEL6 polymorphism at 2019. The KEL polymorphism at 2019 is silent and this allele yields a Js(a-b+) phenotype. Only analysis of the 1910-bp SNP can be used to genotype KEL6 and KEL7.

CD177 polymorphisms: correlation between high-frequency single nucleotide polymorphisms and neutrophil surface protein expression.

BACKGROUND: Human neutrophil antigen-2a (NB1) is located on NB1 glycoprotein, 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 nucleotides. The purpose of this study was to determine if PRV-1 and NB1 are alleles of the same gene or two separate genes; and, moreover, if they are alleles, to explore potential correlations to NB1 glycoprotein expression. STUDY DESIGN AND METHODS: Primer pairs were used to amplify WBC DNA in the regions surrounding the four NB1 polymorphic sites. The four resulting amplicons were sequenced. The size of the neutrophil population in each donor that stained brightly with CD177 antibody was assessed by flow cytometry. RESULTS: If PRV-1 and NB1 are separate genes, then all people should be heterozygous for the PRV-1/NB1 polymorphisms. Because 11 of 23 donors were homozygous for PRV-1 polymorphisms at all four sites, PRV-1 and NB1 are alleles of the same gene, CD177. When the sequenced exons were compared with PRV-1, 13 single nucleotide polymorphisms (SNPs) that result in amino acid changes were found. The G42C NB1 polymorphism, found in 10 donors, was the most common SNP. The size of the CD177 bright-staining neutrophil population was greater in 42C/C donors than in 42G/G donors (66 +/- 20% vs. 41 +/- 21%, p = 0.004). CONCLUSIONS: PRV-1 and NB1 are alleles of the polymorphic gene CD177. The most common SNP in bp 42 predicted an amino acid change that may have an effect on protein expression.

Expression of human neutrophil antigen-2a (NB1) is increased in pregnancy.

BACKGROUND: The expression of human neutrophil antigen-2a (HNA-2a) or NB1 is highly variable. This study investigated variations in neutrophil expression of HNA-2a by comparing the expression of epitopes recognized by three HNA-2a-specific CD177 antibodies among healthy adults and pregnant women. STUDY DESIGN AND METHODS: Flow cytometry was used to compare the neutrophil reactions of three CD177 antibodies (MEM-166, TAG4, and 7D8) among 52 healthy adults. Because HNA-2a expression is greater in women than men, neutrophils were studied from 21 women early in pregnancy and 23 women late in pregnancy. RESULTS: All three CD177 antibodies reacted with two populations of neutrophils: a brightly staining population and a dimly staining population. Among the 52 healthy adults, there was no difference in the mean size of the brightly reactive population of neutrophils recognized by each antibody (46 +/- 23% for MEM-166, 49 +/- 23% for TAG4, and 49 +/- 23% for 7D8), and all three antibodies were specific for HNA-2a. For all three antibodies the size of the antigen bright neutrophil population was greater on neutrophils collected both early and late in pregnancy than in healthy adults. There was no difference in the size of the antigen bright population among neutrophils tested early and late in pregnancy. CONCLUSION: HNA-2a antigen expression increases in pregnancy. Some of the variations in neutrophil expression of HNA-2a among individuals is likely due to differences in gene regulation or differences in post-translational protein modifications rather than gene polymorphisms.