|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 2 (January 15), 1999:
pp. 756-758
CORRESPONDENCE
Flow Cytometric Analysis of Wiskott-Aldrich Syndrome (WAS)
Protein in Lymphocytes From WAS Patients and Their Familial
Carriers
 |
LETTER |
To the Editor:
Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder
characterized by thrombocytopenia with small platelets, severe eczema,
and recurrent infections due to defects in the immune system.1 Recent improvement of the prognosis of WAS by bone marrow transplantation (BMT) made early diagnosis more important. However, early diagnosis is sometimes difficult, because WAS patients often show atypical clinical phenotypes in infancy.2
Although the cause of WAS has been defined as mutations in the WAS
protein (WASP) gene, it is difficult to perform mutation analysis of
the gene in all the possible cases. In this study, we performed flow cytometric analysis of the WASP expression in lymphocytes using an
anti-WASP monoclonal antibody to assess its usefulness in the diagnosis
of WAS. The results suggested that the method is simple, rapid, and
applicable for screening of WAS.
Eight normal individuals, three WAS patients, and their mothers were
included in this study. The diagnosis of WAS was made by their clinical
and laboratory findings and was confirmed by mutation analysis
(Table 1). Restriction enzyme
digestion of polymerase chain reaction (PCR) fragments, allele-specific
PCR, and/or direct sequence analysis of genomic DNA, which were
based on mutation information, were performed for carrier diagnosis as
reported by Ariga et al.3 All of the mothers of the WAS patients were proved to be carriers of WAS (data not shown).
Heparinized blood samples were collected and processed each in a set
consisting of those from a WAS patient, his mother, and an appropriate
normal individual under the same conditions. Samples necessary for more
than 1-day transportation were kept at 4°C after collection.
Peripheral blood mononuclear cells (PBMCs) were isolated by standard
Ficoll-Hypaque gradient centrifugation methods. PBMCs were washed in
phosphate-buffered saline (PBS) containing 1% fetal bovine serum.
Cytofix/Cytoperm solution from CytoStain Kits (Pharmingen, San Diego,
CA) was added to thoroughly suspended 2 × 106 PBMCs in
each tube at 4°C for 20 minutes. After washing twice in Perm/Wash
solution, they were reacted with 1:200 diluted mouse anti-WASP
monoclonal antibody (3F3-A54) or 1:5 diluted mouse IgG1
control (Becton Dickinson, San Jose, CA) at 4°C for 30 minutes and washed twice. They were then reacted with 1:200
diluted fluorescein isothiocyanate (FITC)-conjugated goat
antimouse Igs  and light chains (Biosource, Camarillo,
CA) at 4°C for 30 minutes. Samples, thus processed, were
analyzed on a FACSCalibur (Becton Dickinson). A total of 20,000 events of lymphocytes, which were gated based on forward and side
scatter, were counted. Fluorescence intensity was detected by FL1. M1
area (positive fluorescence) was set in each normal individual stained with mouse IgG1 control so that the proportion of positive cells in the
area was 1%.
As shown in Fig 1 (solid lines), when
compared with normal individuals, deficient WASP expressions were
distinctly demonstrated in all of the WAS patients tested. WASP
expressions in all of their mothers were the same as in those of normal
individuals. The same normal WASP expression was also demonstrated in a
patient with successful BMT (patient no. 1, post-BMT). WASP expressions in the remaining five normal individuals were the same as those of
controls 1, 2, and 3, even when stored at 4°C for 2 days after collection (data not shown). Thus, so far, the method
tested seems to be efficient in the diagnosis of WAS.
View larger version (34K):
[in this window]
[in a new window]
| Fig 1.
Flow cytometric analysis of WASP expression in normal
individuals (control), WAS patients (patient), and their mothers
(mother). Blood samples from controls 1, 2, and 3 and their respective
mothers were collected with the same condition as those of patients no.
1, 2, and 3, respectively. A blood sample from patient no. 1 after BMT
(patient post-BMT) was collected in the separate examination.
|
|
However, what was unexpected in the present study was a finding that
every sample from the patients reacted with anti-WASP antibody
exhibited fluorescence peaks somewhat shifting from those of negative
controls with mouse IgG1 control (dashed lines in Fig 1). The
proportion of cells belonging to M1 area (positive fluorescence) was
calculated to be 2.5%, 21.9%, and 2.2% in patients no. 1, 2, and 3, respectively, and the proportion of positive cells in
patient no. 2 was apparently higher than those in patients no. 1 and 3. As shown in Table 1, the mutation in
patient no. 2 is missense of exon 1, whereas those in patients no. 1 and 3 are nonsense mutation in exon 7 and premature termination in exon 10, respectively. Thus, it is possible to assume that in patient no. 2 some WASP might be expressed and detectable by the present method,
although we could not detect it using Western blot analysis. It might
be due to differences in sensitivity between the two methods, or
unstable WASP in patient no. 2 might be destroyed during the process of
Western blotting, although this is not proven in the present study. On
the other hand, the fluorescence shift seen in patient no. 1 might be
due to binding of the anti-WASP antibody with non-WASP components in
lymphocytes or to thoroughly nonspecific binding, because epitopes of
the anti-WASP antibody, 3F3-A5, reside in a.a. 202-302 of WASP and WASP
of the patient no. 1 is truncated from a.a. 211, being defective in
most parts of the epitope. This might be also applied to
the results observed in patient no. 3. Nevertheless, it is apparent
that the present flow cytometric analysis of the lymphocytes is capable
of differentiating WAS patients from normal or carrier states. Findings
of normal WASP expressions in lymphocytes from carrier mothers were
consistent with nonrandom X-inactivation reported for WAS
carriers.5
The present procedure is similar to that used for detection of BTK, a
cytoplasmic protein deficient in X-linked agammaglobulinemia. Futatani
et al6 reported a limitation of the procedure in which, among 41 cases tested, normal BTK expression was demonstrated in 1 patient with a missense mutation in BTK gene.6 Thus, a similar limitation should exist in the present procedure. Accumulation of data on this procedure together with mutation analysis is being performed in our laboratory.
Masafumi Yamada
Makoto Ohtsu
Ichiro Kobayashi
Nobuaki Kawamura
Kunihiko Kobayashi
Department of Pediatrics
Hokkaido University School of
Medicine
Hokkaido, Japan
Tadashi Ariga
Yukio Sakiyama
Department of Pediatrics
Teine Keijinkai Hospital
Hokkaido,
Japan
David L. Nelson
National Institutes of Health
National Cancer Institute
Metabolism Branch
Bethesda, MD
Satoshi Tsuruta
Department of Allergy
Shizuoka
Children's Hospital
Shizuoka, Japan
Michiya Anakura
Department of Pediatrics
Kohnan Hospital
Hokkaido, Japan
Nobuyoshi Ishikawa
Department of Pediatrics
Kitami Red
Cross Hospital
Hokkaido, Japan
| |
REFERENCES |
1.
Aldrich RA, Steinberg AG, Campbell DC:
Pedigree demonstrating a sex-linked recessive condition characterized by draining ears, eczematoid dermatitis and bloody diarrhea.
Pediatrics
13:133, 1954[Abstract/Free Full Text]
2.
Sullivan KE, Mullen CA, Blaese RM, Winkelstein JA:
A multiinstitutional survey of the Wiskott-Aldrich syndrome.
J Pediatr
125:876, 1994[Medline]
[Order article via Infotrieve]
3.
Ariga T, Yamada M, Sakiyama Y:
Mutation analysis of five Japanese families with Wiskott-Aldrich syndrome and determination of the family members' carrier status using three different methods.
Pediatr Res
41:535, 1997[Medline]
[Order article via Infotrieve]
4.
Stewart DM, Treiber-Held S, Kurman CC:
Studies of the expression of the Wiskott-Aldrich syndrome protein.
J Clin Invest
97:2627, 1996[Medline]
[Order article via Infotrieve]
5.
Wengler G, Gorlin JB, Williamson JM:
Nonrandom inactivation of the X chromosome in early lineage hematopoietic cells in carriers of Wiskott-Aldrich syndrome.
Blood
85:2471, 1995[Abstract/Free Full Text]
6.
Futatani T, Miyawaki T, Tsukada S:
Deficient expression of Bruton's Tyrosine Kinase in monocytes from X-linked agammaglobulinemia as evaluated by a flow cytometric analysis and its clinical application to carrier detection.
Blood
91:595, 1998[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
T. Wada, S. H. Schurman, G. J. Jagadeesh, E. K. Garabedian, D. L. Nelson, and F. Candotti
Multiple patients with revertant mosaicism in a single Wiskott-Aldrich syndrome family
Blood,
September 1, 2004;
104(5):
1270 - 1272.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. S. Lim and K. S.J. Elenitoba-Johnson
The Molecular Pathology of Primary Immunodeficiencies
J. Mol. Diagn.,
May 1, 2004;
6(2):
59 - 83.
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Shinozaki, H. Kanegane, H. Matsukura, R. Sumazaki, M. Tsuchida, M. Makita, Y. Kimoto, R. Kanai, K. Tsumura, T. Kondoh, et al.
Activation-dependent T cell expression of the X-linked lymphoproliferative disease gene product SLAM-associated protein and its assessment for patient detection
Int. Immunol.,
October 1, 2002;
14(10):
1215 - 1223.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Yamaguchi, T. Ariga, M. Yamada, D. L. Nelson, R. Kobayashi, C. Kobayashi, Y. Noguchi, Y. Ito, K. Katamura, Y. Nagatoshi, et al.
Mixed chimera status of 12 patients with Wiskott-Aldrich syndrome (WAS) after hematopoietic stem cell transplantation: evaluation by flow cytometric analysis of intracellular WAS protein expression
Blood,
July 30, 2002;
100(4):
1208 - 1214.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Ariga, T. Kondoh, K. Yamaguchi, M. Yamada, S. Sasaki, D. L. Nelson, H. Ikeda, K. Kobayashi, H. Moriuchi, and Y. Sakiyama
Spontaneous In Vivo Reversion of an Inherited Mutation in the Wiskott-Aldrich Syndrome
J. Immunol.,
April 15, 2001;
166(8):
5245 - 5249.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Yamada, T. Ariga, N. Kawamura, K. Yamaguchi, M. Ohtsu, D. L. Nelson, T. Kondoh, I. Kobayashi, M. Okano, K. Kobayashi, et al.
Determination of Carrier Status for the Wiskott-Aldrich Syndrome by Flow Cytometric Analysis of Wiskott-Aldrich Syndrome Protein Expression in Peripheral Blood Mononuclear Cells
J. Immunol.,
July 15, 2000;
165(2):
1119 - 1122.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Kanegane, K. Nomura, T. Miyawaki, Y. Sasahara, S. Kawai, S. Tsuchiya, G. Murakami, T. Futatani, and H. D. Ochs
X-linked thrombocytopenia identified by flow cytometric demonstration of defective Wiskott-Aldrich syndrome protein in lymphocytes
Blood,
February 1, 2000;
95(3):
1110 - 1111.
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. M Jones and H. B Gaspar
Immunogenetics: changing the face of immunodeficiency
J. Clin. Pathol.,
January 1, 2000;
53(1):
60 - 65.
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Shcherbina, F. S. Rosen, and E. Remold-O'Donnell
WASP Levels in Platelets and Lymphocytes of Wiskott-Aldrich Syndrome Patients Correlate with Cell Dysfunction
J. Immunol.,
December 1, 1999;
163(11):
6314 - 6320.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
