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 Previous Article | Table of Contents
Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 1110-1111
CORRESPONDENCE
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Letter |
To the editor:
X-linked thrombocytopenia identified by flow cytometric
demonstration of defective Wiskott-Aldrich syndrome protein in lymphocytes
X-linked thrombocytopenia (XLT) is characterized by congenital
thrombocytopenia with small-sized platelets and without
immunodeficiency.1 XLT is an allelic variant of Wiskott
Aldrich syndrome (WAS), and both entities are caused by mutations in
the WAS protein (WASP) gene.2 XLT may be misdiagnosed as
chronic idiopathic thrombocytopenic purpura (ITP) in which the
platelets are normal-sized or enlarged. We have recently identified a
5-year-old boy with XLT who had been originally diagnosed as having
chronic ITP. Deficient expression of WASP in lymphocytes from the
patient was demonstrated by flow cytometric analysis using an anti-WASP
monoclonal antibody (MoAb) and the diagnosis of XLT was confirmed
by mutation analysis. Here we demonstrate the usefulness of flow
cytometric analysis in the diagnosis of XLT.
When the patient was 1 year old, he was referred to the Toyama Red
Cross Hospital for the treatment of atopic dermatitis. Thrombocytopenia
(platelet count: 85 × 103/µl) was found by chance,
since he had no bleeding tendency. When he was 3 years old, he
developed a subcutaneous hematoma in the right inguinal region. His
platelet count at that time was 52 × 103/µl and an
increased level of platelet-associated IgG (99.5 ng/107
platelets) was detected. He had neither neutropenia nor anemia, and his
coagulation systems and immunoglobulin levels were normal. Bone marrow
aspirate demonstrated increased numbers of megakaryocytes, but no
abnormal cells, suggesting the diagnosis of ITP. His platelet count
increased transiently following oral predonine and intravenous high-dose immunoglobulin. Because a maternal uncle (age 30 years old)
had thrombocytopenic purpura since early infancy, we
considered the possible diagnosis of XLT.
The demonstration of a mutation affecting the WASP gene is essential
for the diagnosis of XLT.3 However, the genetic analysis is
labor- and time-consuming. We have established a simple flow cytometric
technique to diagnose X-linked agammaglobulinemia (XLA).4 This technique has recently been adapted to analyze WASP in lymphocytes from WAS patients.5 In this report, we demonstrate the
usefulness of a new anti-WASP MoAb and flow cytometry in the diagnosis
of XLT. Anti-WASP MoAb 5A5 was prepared by immunizing mice with the recombinant protein encoded by the N-terminal 1.4 kilo bp obtained with
the pET Trx fusion system 32 (Takara Shuzo, Kyoto, Japan). Peripheral
blood mononuclear cells (PBMC) were prepared by Ficoll-Hypaque gradient
centrifugation from heparinized venous blood, fixed with 4%
paraformaldehyde, and permeabilized with 0.1% Triton-X in
Tris-buffered saline. The cells were incubated with anti-WASP MoAb 5A5
or control IgG2a, washed twice, and incubated with FITC-conjugated goat
anti-mouse IgG2a antibody (Southern Biotechnology Associates Inc,
Birmingham, AL). After washing, the stained cells were analyzed with an
Epics XL flow cytometer (Coulter Corporation, Miami, FL).
As shown in Figure 1A, deficient WASP
expression in lymphocytes was demonstrable in the patient, which was in
marked contrast to intense expression of WASP in lymphocytes from a
healthy individual and the patient's mother. The diagnosis of XLT was
confirmed by immunoblot and genetic analysis. Whole cell lysate of PBMC
were subjected to immunoblot analysis using the anti-WASP MoAb 5A5. WASP protein determined as an approximately 59 kD protein was present
in PBMCs from a healthy control and from the patient's mother (Figure 1B). In contrast, WASP was not detectable in the lysate
of the patient's PBMC. For mutation analysis, total RNA and genomic
DNA were extracted from PBMC by conventional methods, and subjected to
reverse transcriptase-polymerase chain reaction (RT-PCR), as described
previously.6 We found a single base substitution (G1487A)
at the C-terminal end of exon 11, resulting in the skipping of exon 11 or in the skipping of both exons 10 and 11 in the major of the
transcripts, and amino acid substitution (Asp485Asn) in a smaller
portion of the transcript (Figure 2). His mother and sister were found to have 1 mutated and 1 normal alleles, and thus were carriers.
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| Fig 1.
WASP expression in 3 subjects.
(A) Flow cytometric analysis of WASP expression in normal individual
(control), XLT patient (patient), and his mother (mother). The shaded
areas and the dashed line indicate the staining with anti-WASP and
control antibodies, respectively. (B) A Western blot analysis of WASP
protein in the lysates from the 3 subjects above. WASP and  -actin
protein as internal control were determined as approximately 59 kD and
42 kD proteins, respectively.
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| Fig 2.
Schematic representation showing the multiple alterations
in the WASP cDNA of the patient.
Exons are shown in shaded area.
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Flow cytometric analysis of WASP in PBMC is a simple and rapid test for
the diagnosis of XLT and WAS. Although some mutations in the WASP gene
may allow the expression of a normal amount of WASP in lymphocytes, the
flow cytometric analysis is a useful screening test to
differentiate XLT as well as WAS from chronic ITP, when
the patient is male and has early childhood onset.
Hirokazu Kanegane
Keiko Nomura
Toshio Miyawaki
Department of Pediatrics, Faculty of Medicine, Toyama Medical
and, Pharmaceutical University, Toyama, Japan
Yoji Sasahara
Shin Kawai
Shigeru Tsuchiya
Department of Pediatric Oncology, Institute of Development, Aging
and, Cancer, Tohoku University, Sendai, Japan
Gyokei Murakami
Division of Pediatrics, Toyama Red Cross Hospital, Toyama, Japan
Takeshi Futatani
Hans D. Ochs
Department of Pediatrics, University of Washington School of
Medicine,, Seattle, WA
| |
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Nature Genet.
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Futatani T, Miyawaki T, Tsukada S, et al.
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