Blood, 1 June 2002, Vol. 99, No. 11, pp. 4231-4233
BRIEF REPORT
Molecular basis of NB1 (HNA-2a, CD177) deficiency
Karin Kissel,
Steffi Scheffler,
Mohammed Kerowgan, and
Jürgen Bux
From the Institute for Clinical Immunology and
Transfusion Medicine, Giessen, and the Institute for Clinical
Immunology and Transfusion Medicine, Mannheim, Germany.
 |
Abstract |
Alloimmunization to the neutrophil antigen NB1 (HNA-2a, CD177) can
result in immune neutropenia and transfusion-related acute lung injury.
Recently, we were able to elucidate the primary structure of NB1. To
shed light also on the molecular basis of the NB1-negative phenotype,
we studied the neutrophils of 2 women with NB1-specific alloantibodies
for intracellular and extracellular NB1 expression, NB1-specific mRNA
production, and the presence of the NB1 gene. No antibody
binding to neutrophils was observed by immunofluorescence and
immunoblot using a variety of human and monoclonal NB1-specific antibodies. By reverse transcription-polymerase chain reaction with
NB1-specific primers we could not detect NB1 cDNAs without accessory
sequences, which were found to be introns. The NB1 gene was
present in the genome of both patients. Our data indicate that the
NB1-negative phenotype is the result of different off-frame insertions
on RNA level, resulting in NB1 deficiency on neutrophils.
(Blood. 2002;99:4231-4233)
© 2002 by The American Society of Hematology.
| |
Introduction |
NB1 was first described in 1971 as a
neutrophil-specific antigen,1 and it is included as HNA-2a
in the new nomenclature for human neutrophil antigens.2
Recently, the glycoprotein has been clustered as CD 177. NB1 is
expressed on neutrophils of 97% of whites, 95% of African Americans,
and 88% of Japanese.3-9 A special feature of NB1 is its
expression on a neutrophil subpopulation.10,11 Recently,
we elucidated the primary structure of the NB1
glycoprotein.12 A 1614-base pair (bp) cDNA codes for 437 amino acids (aa), of which 416 residues form an NH2-terminal
extracellular protein with 2 cysteine-rich domains, 3 potential
N-linked glycosylation sites, and a short segment including a
glycosyl-phosphatidylinositol (GPI) attachment site. NB1 sequence
differs from deduced PRV-1 protein (polycythemia rubra
vera-1)13 in 4 amino acids.12 Database
searches revealed homology to the Ly-6 (uPAR) domain, suggesting that
NB1 glycoprotein belongs to the uPAR/CD59/Ly-6 snake toxin superfamily.
In this study, we shed light on the molecular basis of the NB1-negative
phenotype by analyzing the neutrophils of 2 women who had formed NB1
alloantibodies, probably during pregnancy. NB1 alloimmunization is
clinically important because NB1 alloantibodies can cause immune
neutropenia and transfusion-related acute lung
injury.1,14-19
| |
Study design |
Sera containing NB1-specific alloantibodies were obtained from
mothers who gave birth to neonates with alloimmune neonatal neutropenia. Approval was obtained from the institutional review board
at University Hospital of Giessen for this study. Informed consent was
provided according to the Declaration of Helsinki. NB1-specific
monoclonal antibodies (mAbs) 7D8, TAG 4, MEM 166, and 1B5 were kindly
provided by Dr D. Stroncek (Bethesda, MD), Dr K. Taniguchi
(Hiroshima, Japan), Dr V. Horesji (Praha, Czech Republic), and Dr L. Clement (Los Angeles, CA). An mAb specific for CD16, 3G8, was purchased
from Immunotech (Marseilles, France). Granulocyte isolation,
phenotyping, solubilization, sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, and immunoblotting were performed as previously
described.20 Antibody binding to granulocytes was
determined by flow cytometry and fluorescence microscopy. Isolation of
granulocyte mRNA and amplification of NB1 3', 5' untranslated regions
and coding region were performed using sense and antisense primers
based on NB1 cDNA sequence and amplification protocol.12
cDNA was subcloned into pGEMT vector (Stratagene, La Jolla, CA),
sequenced according to the manufacturer's protocols, and analyzed
using Internet programs.12 For amplification of genomic
DNA fragments to establish NB1 fragments encompassing exons 5 to 7 (patient 1) and 3 to 4 (patient 2), 5 µL genomic DNA was amplified
with 200 µM of each dNTP, 0.5 µM sense and antisense primers, 4 U
Taq polymerase XL (Perkin Elmer, Weiterstadt, Germany), 15 µL 10×
polymerase chain reaction (PCR) buffer, and 1.25 mM Mg(OAC)2 in a total volume of 50 µL. Initial denaturation
was at 94°C, followed by 35 cycles of 15 seconds at 94°C and 10 minutes at 65°C to 68°C. Final elongation was 10 minutes at 72°C
(GeneAmp Systems 2400; Perkin Elmer). NB1 exon-intron borders were
established by comparison with human genome sequences in GenBank and
experimental data (not shown).
| |
Results and discussion |
On the neutrophils of 2 NB1-alloimmunized women (patients 1 and
2), NB1 glycoprotein was not detectable by immunofluorescence using
NB1-specific mAbs 7D8 and 1B5 or human alloantibodies. The neutrophils
of their siblings, one sister each, were also typed serologically for
NB1 and were found to be NB1
. Testing neutrophil lysates
of the 2 women for the presence of NB1 glycoprotein in immunoblot, none
of the 12 polyclonal or 3 monoclonal (TAG 4, MEM 166, and 7D8) blotting
anti-NB1 antibodies used showed the 50- to 64-kd band typical for NB1
(data not shown). Based on these findings, we analyzed whether
NB1-specific mRNA is synthesized by the neutrophils of the 2 NB1 patients. For that we isolated neutrophil RNA from
the NB1 neutrophils and transcribed it in cDNA, which was
amplified and subcloned. In patient 1, we identified 3 clones among 27 tested containing NB1-specific cDNA. This cDNA (GenBank accession
number AC AJ310433) spans 1717 bp, consisting of a 27-bp 5' coding region, a 946-bp 3'-untranslated coding region, and a 744-bp coding region. In patient 2, we identified 2 clones among 18 tested containing NB1-specific cDNA spanning 1511 bp (AC AJ305326) consisting of a 435-bp
coding region and a 1076-bp 3' untranslated region (Figure
1B). All clones had identical accessory
sequences (specific for each donor) and were of identical length.
Compared with NB1 reference sequence,12 both cDNAs showed
off-frame insertions of 118 bp (position 655-773, patient 1) and 145 bp
(position 381-526, patient 2), respectively. Both insertions showed the
stop codon tga. In patient 1, the predicted protein (Figure 1A)
consisted of 248 amino acids, of which the first 21 form the signal
sequence. The remaining 227 residues code for an NH2-terminal protein,
with one domain homologous to Ly-6 (uPAR) and 2 potential N-linked glycosylation sites. In patient 2, a 145-aa protein can be predicted, consisting of a 21-aa signal peptide and a 124-aa NH2-terminal protein
part containing one Ly-6 (uPAR) homologous domain and one potential
N-linked glycosylation site (Figure 1B). In both patients the deduced
proteins would lack GPI linkage sites and transmembrane segments.
Although the presence of such soluble NB1 glycoprotein fragments, which
are not recognized by the polyclonal and monoclonal antibodies used in
immunoblot, cannot be excluded, these putative fragments do not prevent
NB1 alloimmunization. Thus, our findings indicate that the
NB1 phenotype is the result of common NB1 glycoprotein
deficiency. Analyzing the genomic organization of the NB1
gene in an NB1-expressing patient, we identified 9 exons (Figure
2B). Based on this finding, we analyzed
the genomic DNA sequence encompassing cDNA nucleotides 655-773 (patient 1) and 381-526 (patient 2) in NB1-expressing and
NB1 patients. DNA fragments of 5.8 kbp (patient 1) and
1.3 kbp (patient 2) were amplified (Figure 2A). Length of genomic
fragments from NB1 and NB1-expressing donors were
identical. Sequence analysis of the 4 genomic DNA fragments showed that
the accessory fragments were present in the NB1 gene of
NB1-expressing and -deficient donors. Analysis of the regions
encompassing the dinucleotides gt/ag at the intron termini did not
detect differences between NB1+ and NB1
(patient 2) so that the found off-frame insertions are not caused by
mutations in these splicing sites. Because it is known that the
recognition of splicing sites requires cross-talk between multiple
components with relatively weak interactions,21 it is not
unlikely that NB1 deficiency is the result of incorrect splicing
complex formation. However, this warrants further investigation. Our
results show that in contrast to Fc
RIIIb-deficiency, which is caused
by FcRIIIB gene deletion and leads to the HNA (NA) null
phenotype,22 NB1 deficiency is the result of a gene
expression defect.
View larger version (59K):
[in this window]
[in a new window]
| Figure 1.
cDNA nucleotide sequence of human
NB1 phenotypes and deduced amino acid sequences.
(A) Patient 1 (AC AJ310433). (B) Patient 2 (AC AJ305326). Signal
sequence is shown in bold letters. Potential N-linked glycosylation
sites (*) are indicated. Stop codons are framed. Differences to NB1
reference sequence12 are indicated in italic and bold
letters. Ly-6 (uPAR)-domains comprise aa 131-218 in patient 1 and aa
43-124 in patient 2.
|
|
View larger version (49K):
[in this window]
[in a new window]
| Figure 2.
Amplification of NB1-genomic fragments by PCR and exon
organization of NB1 cDNA.
(A) Primers were numbered according to the NB1 cDNA reference
sequence.12 5.8 kbp fragment: FP1 (409-426) and reverse
primer RP1 (859-879) (lanes 2-4). 1.3 kbp fragment: FP2 (329-348) and
RP 2 (416-436) (lanes 7-9). Lanes 4 and 9: NB1 patient 1 and patient 2, respectively. Lanes 3 and 8: NB1-expressing healthy
persons. Lanes 1 and 6: molecular weight standards. Lanes 2 and 7:
negative controls. (B) Genomic DNA from NB1+ and
NB1 patients was amplified by PCR and sequenced.
Exon-intron borders were determined according to the NB1 cDNA
reference sequence12 and dinucleotides gt/ag at the
intron termini.
|
|
| |
Footnotes |
Submitted August 21, 2001; accepted January 24, 2002.
Supported by a research grant from the Deutsche Forschungsgemeinschaft
DFG BU 770/3-4.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Juergen Bux, Institute for Clinical Immunology and
Transfusion Medicine, Langhansstrasse 7, 35385 Giessen, Germany;
e-mail: juergen.bux{at}immunologie.med.uni-giessen.de.
| |
References |
1.
Lalezari P, Murphy GB, Allen FH.
NB1, a new neutrophil antigen involved in the pathogenesis of neonatal neutropenia.
J Clin Invest.
1971;50:1108-1115[Medline]
[Order article via Infotrieve].
2.
ISBT Working Party on Platelet and Granulocyte Serology.
Nomenclature of granulocyte antigens [abstract].
Vox Sang.
1999;77:251.
3.
Clay ME, Stroncek DF.
Granulocyte immunology. In:
Ness P,Anderson K, eds.
Scientific Basis of Transfusion Medicine. Philadelphia, PA: WB Saunders; 1994:244-280.
4.
McCullough J, Clay M, Press C, Kline W.
Granulocyte Serology: A Clinical Laboratory Guide. Chicago IL: ASCP Press; 1989.
5.
Bux J.
Molecular genetics of granulocyte polymorphisms.
Vox Sang.
2000;78:125-130[Medline]
[Order article via Infotrieve].
6.
Bierling P, Poulet E, Fromont P, Seror T, Bracq C, Duedari N.
Neutrophil-specific antigen and gene frequencies in the French population.
Transfusion.
1990;30:848-849[Medline]
[Order article via Infotrieve].
7.
Lin M, Chen CC, Wang CL, Lee HL.
Frequencies of neutrophil specific antigens among Chinese in Taiwan [abstract].
Vox Sang.
1994;66:247[Medline]
[Order article via Infotrieve].
8.
Matsuo K, Lin A, Procter JL, Clement L, Stroncek D.
Variations in the expression of granulocyte antigen NB1.
Transfusion.
2000;40:654-662[CrossRef][Medline]
[Order article via Infotrieve].
9.
Otho H, Matsuo Y.
Neutrophil-specific antigens and gene frequencies in the Japanese [abstract].
Transfusion.
1989;29:654.
10.
Stroncek DF, Shankar R, Litz C, Clement L.
The expression of NB1 antigen on myeloid precursors and neutrophils from children and umbilical cords.
Transfus Med.
1998;8:119-123[Medline]
[Order article via Infotrieve].
11.
Goldschmeding R, van Dalen CM, Faber N, et al.
Further characterization of the NB1-antigen as a variably expressed 56-62 kD GPI-linked glycoprotein of plasma membranes and specific granules.
Br J Haematol.
1992;81:336-345[Medline]
[Order article via Infotrieve].
12.
Kissel K, Santoso S, Hofmann C, Stroncek D, Bux J.
Molecular basis of the neutrophil glycoprotein NB1 (CD177) involved in the pathogenesis of immune neutropenia and transfusion reactions.
Eur J Immunol.
2001;31:1301-1309[CrossRef][Medline]
[Order article via Infotrieve].
13.
Temerinac S, Klippel S, Strunck E, et al.
Cloning of PRV-1, a novel member of the uPAR receptor superfamily, which is overexpressed in polycythemia rubra vera.
Blood.
2000;95:2569-2576[Abstract/Free Full Text].
14.
Eastlund DT, Charbonneau TT, Steinmetz J.
Granulocyte antibody detection to diagnose immune granulocytopenia and transfusion reaction due to leucocyte incompatibility.
Hematol Rev.
1992;6:201-205.
15.
Stroncek DF, Shapiro RS, Filipovich AH, Plachta LB, Clay ME.
Prolonged neutropenia resulting from antibodies to neutrophil-specific antigen NB1 following marrow transplantation.
Transfusion.
1993;33:158-163[Medline]
[Order article via Infotrieve].
16.
Stroncek DF, Shankar RA, Herr GP.
Quinine-dependent antibodies to neutrophils react with a 60-Kd glycoprotein on which neutrophil-specific antigen NB1 is located and an 85-Kd glycosyl-phosphatidylinositol-linked N-glycosylated plasma membrane glycoprotein.
Blood.
1993;15;81:2758-2766.
17.
Stroncek DF, Herr GP, Maguire RB, Eiber G, Clement LT.
Characterization of the neutrophil molecules identified by quinine-dependent antibodies from two patients.
Transfusion.
1994;34:980-985[CrossRef][Medline]
[Order article via Infotrieve].
18.
Bux J, Becker F, Seeger W, Kilpatrick D, Chapman J, Waters A.
Transfusion-related acute lung injury due to HLA-A2-specific antibodies in recipient and NB1-specific antibodies in donor blood.
Br J Haematol.
1996;93:707-713[CrossRef][Medline]
[Order article via Infotrieve].
19.
Leger R, Palm S, Wulf H, Vosberg A, Neppert J.
Transfusion-related lung injury with leukopenic reaction caused by fresh frozen plasma containing anti-NB1.
Anesthesiology.
1999;91:1529-1532[CrossRef][Medline]
[Order article via Infotrieve].
20.
Bux J, Stein EL, Bierling P, et al.
Characterization of a new alloantigen (SH) on the human neutrophil Fc receptor IIIb.
Blood.
1997;89:1027-1034[Abstract/Free Full Text].
21.
Reed R.
Initial splice-side recognition and pairing during pre-mRNA splicing.
Curr Opin Genet Dev.
1996;6:215-220[CrossRef][Medline]
[Order article via Infotrieve].
22.
Haas de M, Kleijer M, van Zwieten R, Roos D, von dem Borne AEGKr.
Neutrophil FcRIIIb deficiency, nature and clinical consequences: a study of 21 individuals from 14 families.
Blood.
1995;86:2403-2413[Abstract/Free Full Text].
Top
Abstract
Introduction
