The Journal of Immunology, 2000, 165: 1119-1122.
Copyright © 2000 by The American Association of Immunologists
Determination of Carrier Status for the Wiskott-Aldrich Syndrome by Flow Cytometric Analysis of Wiskott-Aldrich Syndrome Protein Expression in Peripheral Blood Mononuclear Cells1
Masafumi Yamada*,
Tadashi Ariga2,
,
Nobuaki Kawamura*,
Koji Yamaguchi,
Makoto Ohtsu*,
David L. Nelson
,
Tatsuro Kondoh§,
Ichiro Kobayashi*,
Motohiko Okano*,
Kunihiko Kobayashi* and
Yukio Sakiyama
*
Department of Pediatrics, Hokkaido University School of Medicine, Sapporo, Japan;
Department of Human Gene Therapy, Hokkaido University School of Medicine, Sapporo, Japan;
National Institutes of Health, National Cancer Institute, Metabolism Branch, Bethesda, MD 20892;
§
Department of Pediatrics, Nagasaki University School of Medicine, Nagasaki, Japan
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Abstract
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The Wiskott-Aldrich syndrome (WAS) is caused by defects in the WAS
protein (WASP) gene on the X chromosome. Previous study disclosed that
flow cytometric analysis of intracellular WASP expression (FCM-WASP
analysis) in lymphocytes was useful for the diagnosis of WAS patients.
Lymphocytes from all WAS patients showed WASPdim instead of
WASPbright. Here we report that FCM-WASP analysis in
monocytes could be a useful tool for the WAS carrier diagnosis.
Monocytes from all nine WAS carriers showed varied population of
WASPdim together with WASPbright. None of
control individuals possessed the WASPdim population. In
contrast, lymphocytes from all the carriers except two lacked the
WASPdim population. The difference of the
WASPdim population in monocytes and lymphocytes observed in
WAS carriers suggests that WASP plays a more critical role in the
development of lymphocytes than in that of monocytes. The present
studies suggest that a skewed X-chromosomal inactivation pattern
observed in WAS carrier peripheral blood cells is not fixed at the
hemopoietic stem cell level but progresses after the lineage
commitment.
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Introduction
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The
Wiskott-Aldrich syndrome
(WAS)3 is an X-linked
recessive disorder characterized by severe recurrent infections due to
immunodeficiency, tendency to bleeding due to thrombocytopenia with
small platelets, and severe eczema (1, 2). The responsible
WAS protein (WASP) gene was recently identified (3), and
since then a lot of mutations in this gene have been reported with
defective or reduced expression of WASP (4, 5). WASP is
preferentially expressed in hemopoietic cells, such as lymphocytes,
monocytes, and platelets (6). Although the definite WASP
function remains to be determined, WASP seems to play roles in signal
transduction pathways for cell growth (7) and in
cytoskeletal organization in response to activation
(8).
In a previous study, we have established the technique of flow
cytometric analysis of intracellular WASP expression (FCM-WASP
analysis) in lymphocytes (9) and demonstrated its clinical
usefulness for the diagnosis of WAS patients. However, the results
suggested that application of this technique for the carrier diagnosis
is not possible because all the three carriers studied had only the
WASPbright population of lymphocytes.
In this study, we used monocytes for FCM-WASP analysis, and found that
all the nine carriers studied had both the
WASPbright and WASPdim
population in varying degree. We conclude that this analysis could be a
useful tool for the WAS carrier diagnosis. The mechanism of the skewed
X-chromosomal inactivation pattern in WAS carriers was discussed, based
on the difference of the WASPdim population
between monocytes and lymphocytes observed in WAS carriers.
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Materials and Methods
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Carriers studied
Nine WAS carrier mothers from different families and a WAS
patient, who had received bone marrow transplantation (BMT) from his
carrier mother, were included in this study. One noncarrier mother of a
WAS patient was also studied. The results of mutations in the WASP gene
belonging to each family, some of which were reported elsewhere
(10, 11), are shown in Table I
. Carrier diagnosis was
performed by molecular methods as described previously
(10). Carrier mothers in families 1 and 3 are the mothers
of patients 1 and 2 in the previous study (9). Carrier
mothers in families 3 and 4 are unrelated but had the same mutated
allele. BMT was performed in the patient in family 5 from his mother 3
years previously. The severity of WAS-associated symptoms of patients
in all the families was scored from 1 to 5, based on the criteria by
Zhu et al. (12).
FCM-WASP analysis
FCM-WASP analysis was performed as previously described
(9) with minor modification. In brief, PBMC washed in PBS
containing 1% FBS were treated with Cytofix/Cytoperm solution from
CytoStain kit (PharMingen, San Diego, CA) at 4°C for 20 min. After
washing twice with Perm/Wash solution, they were reacted with 1:200
diluted mouse anti-WASP mAb (3F3-A5) (6) or 1:5
diluted mouse IgG1 Ab (Becton Dickinson, San Jose, CA) at 4°C for 30
min. They were then reacted with FITC-labeled goat anti-mouse IgG1
Ab (Southern Biotechnology Associates, Birmingham, AL). Samples, thus
processed, were analyzed on a FACSCalibur (Becton Dickinson, Mountain
View, CA). Gating of lymphocytes or monocytes was made from a
distribution pattern in forward and side scatter. A total of 20,000
events of each cell were studied.
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Results
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Significant difference of WASP expression in monocytes between
normal individuals and WAS patients
As we have shown the significant difference of WASP expression in
lymphocytes previously (9), the same difference of WASP
expression in monocytes was observed between normal
individuals and WAS patients. An example of a normal individual
(A, WASPbright) and a WAS patient from
family 3 (B, WASPdim) was shown (Fig. 1).
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FIGURE 1. FCM-WASP analysis in monocytes from WAS patients. Results of a normal
individual (A) and a WAS patient in family 3
(B) are shown. The WASP expression was significantly
reduced (WASPdim) in the patient compared with that of the
normal individual. The same results were obtained from patients in
other families.
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WAS carriers possessed the WASPdim population in
monocytes
In all the carriers, the WASPdim population
was detected in monocytes in variable degree with the major population
of WASPbright. The pattern of the
WASPdim population in WAS carriers was largely
divided into three groups according to the proportion of
WASPdim cells: skewed (A, <10%),
moderately skewed (B, >10%, <30%), and random
(C, >30%) groups (Fig. 2,
A–C). Carriers in families 1 and 2 belong to group A,
carriers in families 3, 4, 5, 6, and 10, and a patient in family 5
after BMT to group B, and carriers in families 7 and 8 to group C. The
proportion of the WASPdim population ranged from
3.5% to 50.7% (Table I
). None of
control individuals including a noncarrier mother in family 9 possessed
the WASPdim population (Fig. 2D).
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FIGURE 2. FCM-WASP analysis in monocytes from three WAS carriers and one
noncarrier. The pattern was largely divided into three groups according
to the proportion of WASPdim cells: skewed
(A, <10%), moderately skewed (B,
>10%, <30%), and random (C, >30%) groups.
A, B, and C are the
results of carriers in families 1, 6, and 8, respectively. The
proportion of cells in M1 area was shown in Table I as the percentage
of WASPdim cells. The results of a noncarrier mother of
family 9 were also shown (D).
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The WASPdim population in lymphocytes was not detected
in most of WAS carriers
In contrast to FCM-WASP analysis in monocytes, most of carriers
(seven of nine) were shown to have only the
WASPbright population of lymphocytes (Fig. 3 and Table I). The
WASPdim population of lymphocytes was detected
only in carriers in families 7 and 8 (the data of a carrier in family 8
is shown in Fig. 3C).
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FIGURE 3. FCM-WASP analysis in lymphocytes. Results of a normal individual
(A), a patient of family 8 (B), and a
carrier in family 8 (C) are shown. Most of carriers
(seven of nine) were shown to have only the WASPbright
population of lymphocytes. The WASPdim population was
demonstrated in lymphocytes from a carrier in family 8. The proportion
of cells in M1 area was shown in Table I as the percentage of
WASPdim cells.
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Discussion
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In this study, we report that FCM-WASP analysis in monocytes could
be a useful tool for WAS carrier diagnosis. All the definitely
diagnosed WAS carriers possessed the WASPdim
population of monocytes together with the
WASPbright one. The proportion of the
WASPdim population ranged from 3.5% to 50.7%
and could be detected clearly.
In the previous study, we have established the technique of FCM-WASP
analysis in lymphocytes (9) and demonstrated its clinical
usefulness for the diagnosis of WAS patients. However, the application
of this technique for the carrier diagnosis seemed not to be possible
because all the three carriers studied had only the
WASPbright population of lymphocytes. We could
not find any difference between WAS carriers and control females in
these studies. This finding is not surprising, because a skewed
X-chromosomal inactivation pattern is observed in female carriers of
some X-linked genetic disorders including WAS in constitutional or in
some specific lineage cells. The cause of this phenomenon is
attributable to negative selection of cells, which have the growth
disadvantage due to the mutant gene on the active X chromosome
(13). Wengler et al. further reported that a skewed
X-chromosomal inactivation pattern in WAS-obligate carriers occurs
early during the hemopoietic differentiation, speculating that
hemopoietic progenitors are affected by WASP mutations
(14). In contrast, a random X-chromosomal inactivation
pattern was reported in some carriers, although these reports are
limited to carriers of X-linked thrombocytopenia or mild WAS with
missense mutations (15).
In the present study, we performed FCM-WASP analysis in PBMC to study
whether a skewed X-chromosomal inactivation pattern is universally
observed in all hematological cells of WAS carriers. The results showed
that all the WAS carriers have variable proportion of
WASPdim population of monocytes, together with
the WASPbright one. We then quantitatively
evaluated the X-chromosomal inactivation pattern in monocytes from each
carrier. The WASPdim monocyte population detected
in carriers in families 1 and 2, whose WASP gene mutation seemed to
result in nearly null function of WASP, was 5.4% and 3.5%,
respectively, whereas carriers in families 7 and 8 who have the
missense mutations were revealed to have the
WASPdim population of 30.1% and 50.7%,
respectively. These results may suggest the proportion of the
WASPdim monocyte population correlates with their
genotypes. However, a carrier in family 5 who was supposed to have the
most severe mutation, and a carrier in family 6 with missense mutation,
both belong to the same B group of moderately skewed pattern (the
WASPdim population of 17.3% and 11.7%,
respectively). Thus, genetic, stochastic, or other unknown factors
might contribute to the skewed pattern in monocytes as well as types of
WASP gene mutations.
In contrast to the observation in monocytes, we could not detect the
WASPdim population of lymphocytes in seven of
nine carriers. Only two carriers with random X-inactivation pattern in
monocytes were shown to have the WASPdim
population of lymphocytes. Even in the two carriers, the
WASPdim population of lymphocytes was smaller
than those of monocytes were. Thus, WASP seemed to be more critical for
the development of lymphocytes than that of monocytes. Alternatively,
accelerated cell death in WASPdim lymphocytes by
spontaneous apoptosis (16) might be responsible for the
lack of WASPdim lymphocytes in most of WAS
carriers. We speculate that the mutant WASP in these two families still
remains to have a residual function that makes the carriers’
WASPdim lymphocytes survive in their peripheral
blood.
Although FCM-WASP analysis in monocytes is much simpler and faster than
molecular methods for WAS carrier diagnosis, it is possible that some
carriers will have undetectable percentages of
WASPdim monocytes. Therefore, we should carefully
rule out the possibility of carriers when the
WASPdim population was not detected. And as was
discussed for the diagnosis of WAS in the previous study
(9), this method of carrier detection should be also
limited to families where the affected boys have a
WASPdim population, although a normal amount of
WASP expression has not been reported in WAS patients.
We had a chance to study the WAS patient (patient in family 5) who had
received BMT from his carrier mother (carrier in family 5) 3 years
previously. He was shown to have almost the same proportion of the
WASPdim population of monocytes as his mother
(18.5% vs 17.3%). Also similar to his mother, he did not have
WASPdim lymphocytes, either. Because the
karyotype of the patient had completely changed to 46, XX after BMT,
both lymphocytes and monocytes analyzed were originated from the
hematological progenitor cells of the carrier mother. The results
indicate that transplantable hemopoietic progenitor cells in the
carrier mother was not severely skewed, and the proportion of the
reconstructed WASPdim monocyte population was
reproducible in vivo.
We previously demonstrated that most of the WAS carriers possessed
lymphocytes and granulocytes expressing the mutant WASP message by
allele-specific RT-PCR methods (17). These results were
partly inconsistent with the present results, because seven of nine
carriers did not have the WASPdim lymphocyte
population in this study. In the previous study, however, we did not
separate monocytes from the lymphocyte fraction. Therefore, it was
possibly monocytes that predominantly expressed the mutant WASP message
because "lymphocytes" as described in the previous report included
lymphocytes and monocytes. If it is the case, the previous results
obtained by RT-PCR methods mostly agree with those by flow cytometric
analysis in this study.
In conclusion, this study demonstrated the usefulness of FCM-WASP
analysis in monocytes for the carrier diagnosis of WAS. The difference
of the WASPdim population between in monocytes
and lymphocytes from WAS carriers suggests that a skewed X-chromosomal
inactivation pattern observed in WAS carriers’ blood cells is not
fixed at the hemopoietic stem cell level but progresses after the
lineage commitment.
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Acknowledgments
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We thank Drs. Mika Iwamura, Hirotaka Takahashi, Nobuyoshi Ishikawa,
Tsuyosi Ito, and Michiya Anakura for allowing us to analyze their
patients and their families.
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Footnotes
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1 This work was supported by a Health Science Research Grant from the Ministry of Health and Welfare of Japan. 
2 Address correspondence and reprint requests to Dr. Tadashi Ariga, Department of Human Gene Therapy, Hokkaido University School of Medicine, N-14 W-5, Kita-ku, Sapporo, 060-8638, Japan.
3 Abbreviations used in this manuscript: WAS, Wiskott-Aldrich syndrome; WASP, WAS protein; FCM-WASP, flow cytometric analysis of WASP expression; BMT, bone marrow transplantation.
Received for publication February 22, 2000.
Accepted for publication April 26, 2000.
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Abstract
Introduction
