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The Center for Blood Research and the Department of Pediatrics, Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mutated gene responsible for the disorder was identified in 1994 together with its protein product, WASP (Wiskott-Aldrich syndrome protein) (4), a 53-kDa intracellular hemopoietic cell protein (4, 5, 6, 7). Subsequent biochemical studies characterized WASP as a multidomain molecule that appears capable of regulating the actin cytoskeleton, findings that resonate well with the defective cytoarchitecture of WAS patient blood lymphocytes (8, 9, 10) and their defective responsiveness to select stimuli (10, 11). Individual WASP domains bind phosphoinositol 4',5' bisphosphate (PIP2) (12); WIP (WASP interacting protein), an actin-binding protein (13); cdc42, a GTPase that regulates filopodial and lamellipodial surface extensions (14, 15, 16); and Src homology (SH) 3-containing proteins including Grb2 and Fyn (Refs. 17 and 18 , and references therein). WASP also contains a cofilin-like domain and a verprolin domain (12). Altogether, these findings hold promise that an integrated picture of normal WASP function will emerge in the near future, and with it, a better understanding of the pathological events in WAS patient cells.
On the other hand, certain findings are unexplained in current models. For example, WASP is expressed in all non-erythroid hemopoietic cells examined, including T cells, B cells, NK cells, monocytes (4, 5, 6, 7), and neutrophils (this study), and indeed normal WASP levels are similar for many of these cells (this study), yet the pathology of the disease is primarily ascribed to platelets and T lymphocytes (reviewed in Refs. 3 and 18). Another unexplained feature is the variability of clinical symptoms. Whereas platelet defects are present from birth and are severe in all patients, the immune defects are extremely variable, both in age of onset and severity, ranging from negligible, in which case the disease has been also called X-linked thrombocytopenia, to life threatening.
If newly diagnosed WAS infants are to benefit from current and future therapeutic modalities, it is important that the basis of disease severity be understood. The present manuscript explores the premise that WASP expression levels in patients’ cells are an important determining factor for clinical outcome. The study takes advantage of WASP expression data established for EBV-transformed cell lines of a diverse patient panel (19). Fresh blood samples were obtained from these and additional patients, and WASP levels were separately quantified for lymphoid cells, platelets, and neutrophils and compared with established levels in EBV cell lines.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Patients were diagnosed with the WAS based on male sex, thrombocytopenia with small platelets, eczema, immunodeficiency of variable clinical severity, and, in some cases, family history. WASP mutations for the patient donors of EBV cell lines P31-P35 and P37-P44 were previously described (19, 20, 21). Mutation identification for patients TS, OW, and WR was provided by Dr. Hans Ochs (University of Washington School of Medicine, Seattle WA) (unpublished data), for patients MR and LD by Dr. Sau-Ping Kwan (Rush Medical School, Chicago, IL) (unpublished data), for the kindred TM and SM by Dr. Alfons Meindl, Pediatric Clinic, University of Munich, Munich, Germany (unpublished data), and for patient AA by Drs. Silvia Giliani and Luigi Notarangelo, Department of Pediatrics, University of Brescia, Brescia, Italy (unpublished data). Mutations for patients NK and SV were identified by sequencing amplified exons of blood cell DNA using a modified strategy (L. Jones and E. Remold-O’Donnell, unpublished observations).
Blood cells
Paired blood samples from WAS patients and normal healthy consenting individuals were collected in acid-citrate-dextrose (ACD; NIH formula A) and fractionated immediately or after overnight shipment at ambient temperature. The blood was centrifuged at 200 x g for 12 min to separate platelet-rich plasma (PRP) and pelleted cells. Additional ACD were added (1 part per 3 parts PRP), and platelets were pelleted at 800 x g for 15 min. The platelets were resuspended in 10 mM TES buffer (pH 7.2), 136 mM NaCl, 2.6 mM KCl, 0.5 mM NaH2P04, 2 mM MgCl2, 0.1% glucose, and 0.1% BSA; additional ACD (20%) was added, and prostacyclin (1 µg/ml) and the platelets were pelleted at 800 x g for 10 min.
Pelleted blood cells (after removal of PRP) were combined with equal
volume 2% dextran in 150 mM saline for 30–40 min at 
22°C to
sediment erythrocytes. The supernatant was aspirated, and the
leukocytes were fractionated by Histopaque 1077 centrifugation. PBMC
were collected from the interface layer and pelleted and washed with
HBSS without Ca2+ and Mg2+
by pelleting at 200 x g for 15 min. Neutrophil pellet
was washed with HBSS without Ca2+ and
Mg2+ by pelleting at 200 x g for
15 min, and residual erythrocytes were removed by water lysis.
T and B lymphocytes were immunomagnetically isolated from fresh or frozen WAS patient and normal PBMC, and from frozen WAS patient or control spleen cells. The cells were preincubated at 4°C for 15 min in RPMI 1640 with 10% FCS. Following the manufacturer’s instructions, CD19 magnetic beads (Dynal, Lake Success, NY) were added, and the cell suspension was incubated on a rotator at 4°C for 60 min. The CD19 beads were magnetically collected, washed with cold media, and incubated with Detach-a-Bead (Dynal) overnight at 4°C to release B cells. CD4+ T cells were isolated from the depleted cell suspension by adherence to CD4 immunobeads using the same protocol.
To harvest erythrocytes, pelleted blood cells remaining after removal of PRP and removal of the top half of the cell pellet were suspended in 10 vol of HBSS without Ca2+ and Mg2+ and washed by several cycles of centrifugation for 10 min at 1200 rpm.
Cell lines
EBV-transformed cell lines from WAS patients and normal individuals (19) were grown in RPMI 1640 with 10% FCS, penicillin, and streptomycin. HeLa epithelial carcinoma cells strain S3 were grown as adherent cells in DMEM (high glucose), 10% FCS, penicillin, and streptomycin and were detached for harvest by 10 min incubation in 25 mM EDTA in PBS at 37°C, washed, and lysed.
SDS electrophoresis
Cells were washed at 22°C in
Ca2+/Mg2+-free HBSS
containing 2 mM disopropylfluorophosphate and 25 µg/ml leupeptin.
Lysates were prepared of nucleated cells (1.5 x
107/ml), erythrocytes (7 x
108/ml), or platelets (5 x
108/ml) by suspending the cells in 0.5 vol of
buffer with protease inhibitors (10 mM Tris-HCl (pH 7.4), 150 mM NaCl,
2 mM EGTA, 2 mM disopropylfluorophosphate, and 50 µg/ml leupeptin),
adding 0.5 vol of 4% SDS, 125 mM Tris-HCl (pH 6.8), 4%
mercaptoethanol, 20% glycerol, 50 µg/ml bromphenol blue, and
pipetting for several minutes at 100°C. The cell lysates were stored
at -80°C in aliquots and fractionated by SDS electrophoresis on 9%
polyacrylamide gels (14 cm x 10 cm x 1.5 mm) for Western
blot (described below); one aliquot was stained with Coomassie blue to
verify successful recovery and lysis of cells.
Western blots
SDS gels were transferred at 80 mA for 16 h to
nitrocellulose (or polyvinylidene difluoride (PVDF)) membrane, which
was blocked at 22°C with 2% normal goat serum in PBS, 0.05%
Tween 20 (PBS-Tween). The blots were incubated as described
(19) with affinity-purified rabbit Abs to WASP aa 485–502
(W-485 Ab; 100 ng/ml) for 2 h in PBS-Tween-3% milk, washed, and
incubated for 1 h in PBS-Tween-3% milk containing
125I-labeled goat Abs (0.3 µg/ml) to rabbit
IgG. The WASP bands were quantified using the Storm 860 Imager and
Image Quant v 1.1 program (Molecular Dynamics, Sunnyvale, CA).
Linearity of the immunoblot responses was previously demonstrated
(19).
Protein quantitation
Protein in cell lysates was quantified with Nano-Orange (N-6666 kits; Molecular Probes, Eugene, OR) using bovine albumin as standard. Fluorescence was measured with excitation at 485 nm and emission at 590 nm.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Isolated peripheral blood cells and EBV-transformed B cell lines
of normal individuals were lysed in the presence of protease inhibitors
and examined for WASP content by quantitative Western blot (Fig. 1
A). Because these cells
differ in size, WASP levels are compared on a "per mg of total cell
protein," intended as an approximation of intracellular volume. With
the content of normal PBMC set as 1.0 unit WASP/mg protein, 0.86 units
per mg was found in platelets, 0.78 in
neutrophils,5 and 0.93
units in EBV cell lines (Fig. 1B). WASP was nondetectable in
erythrocytes and in HeLa cells, a nonhemopoietic line (Fig. 1). When
cells from seven normal donors were compared, WASP levels showed only
minor variation, ±7% in PBMC, ±8% in platelets, and ±8% in
neutrophils (5 donors). Mean WASP levels in normal peripheral T
lymphocytes (CD4+) and B lymphocytes
(CD19+) were 0.99 and 0.80 U/mg, respectively
(presented below).
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When PBMC of 25 WAS patients were examined, seven patients were
identified whose PBMC and EBV cell lines are WASP-negative (Table I). All of these patients have severe
disease. For reasons clarified below, the WASP-negative mutations are
called group D.
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Seventeen of 25 patients had WASP-positive PBMC. Two surprising
features were noted. First, WASP expression levels in PBMC varied only
within a narrow range, 7% to 20% of normal levels. Second, WASP
levels in PBMC were in many cases substantially different (both higher
and lower) than the levels in EBV cell lines from the same patients. On
further inspection of the data, distinct patterns were discerned based
on the ratio of WASPEBVL to
WASPPBMC, and the patient data were grouped to
reflect these expression patterns (Fig. 2).
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, upper panel).
Although it is not the basis for the grouping, all identified group A
patients have exon 1 and 2 missense mutations, and most have mild
disease (8 of 9 patients).
In group B (2 patients), WASP levels are comparable in PBMC and EBV
cell lines (Fig. 2, middle panel). The group B patients (one
kindred) have an exon 10 frameshift mutation and moderate disease.
In group C (7 patients), WASPPBMC levels are
7–16% of normal; however, EBV cell lines from these patients do not
express WASP (Fig. 2, lowest panel). Mutations in this group
are diverse, as is disease severity (see Discussion).
The four expression patterns for patient lymphoid cells, three with
WASP-positive PBMC and the WASP-negative group, are summarized
graphically in Fig. 3. We hypothesize
that the different WASP levels in EBV cells and PBMC of group A
patients result from (approximately) normal synthesis coupled with
aberrantly increased degradation. This putative scenario is suggested
also by the high levels of mature size WASP RNA in group A cells, 87%,
86%, 69%, and 85% of normal for EBV cell lines P31, P32, P41, and
P44 cells, respectively, in contrast to the modest levels, 10% of
normal, in the group B cell line (19).
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To explain the group C pattern of WASP-positive PBMC and WASP-negative EBV cell lines, two hypotheses were considered. First, expression vs non-expression of mutated WASP might be determined by environmental forces acting on cells, and such forces would be expected to differ for transformed cells in culture compared with primary cells in circulating blood. Alternatively, the negativity of the EBV cell lines might reflect an intrinsic inability of B lymphoid cells to express WASP with group C mutations. In the latter case, peripheral blood B cells of group C patients should also be WASP negative.
To test the latter hypothesis, we separately evaluated
CD4+ cells (T cells) and
CD19+ cells (B cells) isolated by immunomagnetic
purification from patient blood. For normal individuals, the levels of
WASP are 0.99 ± 0.07 U/mg protein in T cells
(CD4+ cells) and 0.80 ± 0.07 U/mg protein
in B cells (n = 5). When WASP levels in isolated
patient T and B cells were each expressed as a percentage of the
corresponding normal population, no significant difference was found
between the T cell level and the B cell level for group A patient P32,
or group A patient P50, or group B patient P34 (Fig. 4). On the other hand, five group C
patients with four different mutations had readily detectable WASP
levels in their isolated CD4+ T cells (6–13% of
normal levels), but no detectable WASP in their isolated B cells (Fig. 4). These findings demonstrate that peripheral blood B lymphoid cells,
like EBV-transformed B cell lines, are unable to express
WASP with group C mutations.
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Patients who have WASP-negative PBMC were found to have WASP-negative neutrophils (5 patients), and most patients with WASP-positive PBMC were found to have WASP at slightly lower levels in neutrophils. The latter pattern was found for eight group A patients (mean of 12.4% in PBMC, 8.6% in neutrophils) and six group C patients (mean of 11.0% in PBMC, 4.9% in neutrophils) (P33 and P39 mutations). However, the group B kindred with mean WASPPBMC of 12% have WASP-negative neutrophils, and the P54 group C kindred with mean WASPT cells of 15% also have WASP-negative neutrophils. Thus, the pattern of expression of mutated WASP genes in neutrophils is dissimilar to both T cells and B cells, i.e., does not correlate with the mutation grouping established here for lymphoid cells.
WASP levels in patient platelets
Isolated platelets in sufficient numbers for WASP assay were
obtained from 18 patients. Included were splenectomized
(n = 14) and eusplenic (n = 4) patients
as well as patients with WASP-positive PBMC from group A
(n = 6), B (n = 2), and C
(n = 7), and also group D (WASP-negative PBMC)
(n = 3). Platelets from all of these patients are WASP
negative (Table II). This finding
strongly suggests that WASP absence in patient platelets contributes
to, or accounts for, the uniform severity of the platelet defect is
this disease.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The finding of WASP absence in platelets of 18 patients, ages 3 years to 43 years, 14 splenectomized and 4 eusplenic, with platelet counts from 15,000 to 250,000 (platelets/µl), and representing 12 mutations and 4 lymphoid cell expression patterns, strongly suggests that WASP negativity of platelets is a universal feature of the disease. Absence of WASP in patient platelets appears to provide a molecular explanation for the severity and consistency of the platelet defect in the WASP.
Findings in the present study clearly demonstrate that different cell
lineages can differ in their ability to handle the same mutated
WASP gene. T cells and B cells are discordant for expression
of P42, P39, P33, and P54 WASP (Figs. 4 and 5). Expression by neutrophils of
WASP mutants correlates in most cases with expression by T
cells; however, T cells and neutrophils are discordant in handling of
the P34 and P54 mutations (Fig. 5). The phenomenon of cell lineage
discordancy in expression of mutant genes, an epigenetic phenomenon, is
unlikely to be limited to WASP. The mechanisms are largely
unknown.
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Alternatively, mutant WASP might be present in megakaryocytes and depleted in platelets due to enhanced proteolysis. Proteolytic depletion of platelet WASP could occur without depletion of megakaryocyte and lymphoid cell WASP because platelets have different proteases and/or because the minimal levels of protein synthesis (protein replacement) in platelets would be permissive for proteolytic depletion. In a previously described case of cell lineage discordancy in expression of a mutant gene, a subset of adenosine deaminase deficiency patients express the enzyme at undetectable or very low levels in erythrocytes, but at much higher levels in lymphocytes; this discordancy was attributed to the degradation of unstable mutated adenosine deaminase, which depletes the enzyme in cells, such as erythrocytes, that cannot synthesize new protein (24). For the putative case of mutated platelet WASP, calpain (Ca2+-dependent neutral protease) should be considered because its levels are high in platelets (25). Also, normal WASP is cleaved when platelet calpain is activated (26), and evidence indicates that WAS platelets have enhanced calpain activation (27). On the other hand, other sensitive calpain substrates, talin and actin binding protein, are not depleted in WAS platelets (27). Another putative mechanism for the absence of WASP in patient platelets is the possible failure of mutated WASP molecules to partition into platelets when the latter bud from megakaryocytes. This hypothesis is consistent with the postulate that megakaryocytes are more sensitive than other hemopoietic cells to loss of WASP (28) and with studies of a megakaryoblastic cell line, which strongly suggest a functional role for WASP in megakaryocyte differentiation (29).
The finding of WASP negativity in the severely dysfunctional patient platelets supports the hypothesis that WASP levels in lymphocytes are also important in determining disease severity. Overall, the present findings support an inverse correlation of WASP lymphoid cell levels and severity of immune dysfunction in most cases. For example, all of the seven WASP-negative patients have severe disease, and the patients with WASP-positive lymphocytes (groups A and B) have mild or moderate disease.
The patients categorized as group A based on their lymphoid cell WASP expression patterns all have exon 1 and 2 missense mutations that map to the PH1 (pleckstrin homology 1) domain (12), which has been previously associated with milder disease (Refs. 7 and 28 , and reviewed in Ref. 30). We hypothesize that enhanced posttranslational proteolysis accounts for the decreased WASP levels in group A PBMC. The different WASPPBMC and WASPEBVL levels for individual group A mutations may reflect different ratios of synthesis to proteolysis in PBMC and proliferating cell lines.
Evidence indicates that transcriptional or translational events are responsible for the decreased or absent WASP levels in most other mutations. Of the group D WASP-negative mutations, P35, an exon 2 nonsense mutation, and P40, an exon 3 frameshift, have no detectable mature RNA, indicating that their mutated WASP genes are not transcribed.
The group B P34 cell line has substantially decreased levels (10% of normal) of mature size RNA, strongly suggesting that the similarly decreased WASP protein levels are due to limiting events at the transcriptional level.
Most surprising are the group C mutations. These diverse mutations,
donor splice site mutations in introns 6 and 7, an intron 6 acceptor
site mutation, and an exon 1 nonsense mutation, were grouped initially
based on discordant WASP expression in PBMC (positive) and EBV cell
lines (negative). Additional experiments showed that the four mutations
share discordant WASP expression at the level of peripheral blood
cells, i.e., positive peripheral T cells and negative peripheral B
cells (Fig. 4). Thus, WASP presence in group C PBMC is due to its
presence in cells other than B lymphocytes.
RT-PCR of PBMC of a group C patient with the P39 mutation (intron 6,
+5, g
a) revealed low levels of multiple RNA species, and sequencing
showed that 30% of the cloned products were without defect
(7). This finding indicates that T cells correctly splice
some P39 pre-RNA despite the intron 6 donor site mutation. The finding,
together with absence of protein and mature size RNA in P39 EBV cells
(19), suggests that T cells and B cells differ in the
stringency of RNA splicing requirements. For a group C patient with the
P33 mutation (intron 6, -1, ga), it is likely that the significant
discordant event in T cells and B cells is also at the transcriptional
level because the patient’s B cell line lacks WASP RNA
(19). RT-PCR of this patient’s PBMC showed multiple RNA
species at low levels (L. Jones and E. Remold-O’Donnell, unpublished
observations), suggesting that P33 T cells produce (some) mature WASP
RNA despite the acceptor site mutation, either by low level use of the
mutated splice site or by the use of an alternative splice site(s). The
third group C splice site mutation (P54), intron 7, +5, ga, has not
been further studied.
In cells of the group C P42 patients (exon 1,
Arg13 stop), the discordant event responsible
for WASP expression in T cells and non-expression in B
cells, is apparently at the translational level because the P42 B cell
line, which lacks WASP protein, has near normal levels of mature size
WASP RNA (19). Inspection of the mutated gene sequence
suggests that synthesis of WASP by P42 T cells occurs by initiation (or
reinitiation) at an internal AUG codon downstream of the mutant stop
codon, possibly at the Met38 codon. Thus, the
discordant mechanisms that allow T cells, but not B cells, to generate
WASP protein from group C mutated genes appear to be variable, which,
if correct, hints at an overall greater stringency of gene expression
mechanisms in B cells compared with T cells.
In terms of disease severity, group C patients are diverse. When seen as young children, the two P42 patients were categorized as severe, the P33 patient as moderate, and most of the P39 patients and P54 patients as mild phenotype (formerly called X-linked thrombocytopenia). This diversity is not surprising because, although all group C patients express some levels of WASP in their T cells, P42-WASP and probably P33-WASP are defective proteins, whereas P39-WASP protein may be normal.
In an apparently unfortunate feature of group C mutations, like group D, the clinical picture seems to be that, with time, a number of the patients develop B cell lymphomas, including one of the two P42 patients (the second underwent early bone marrow transplant), the P33 patient, and members of two of the three P39 kindred described here. It is thus possible that the increased incidence of B cell malignancies in the WAS (31, 32, 33) includes disproportionate numbers of patients with pattern C mutations or patients with WASP-negative B cells (groups C and D). Additional data are needed to test this postulate and, if confirmed, to unravel the underlying mechanism.
Although there are four lymphoid cell expression patterns, the absolute WASP levels in patient PBMC varied only over the range 0 to 20%. Assuming that the patients studied here are representative and that WASP is not secondarily degraded in another disorder, assay of WASP levels in PBMC could be used as a criterion for diagnosis because patient levels would be readily distinguishable from normal. This suggestion has been made previously (e.g., Refs. 34 and 35) and, indeed, a flow cytometric method appropriate for clinical assay of WASP was recently developed (35). The present data on 27 patients provides substantial justification for clinical assay of WASP as part of diagnosis.
WASP levels have been assayed for EBV cell lines of >60 patients
(5, 7, 19, 34), but levels in PBMC have been previously
determined for only 15 patients (7, 34). We note, and
cannot completely explain, the discrepancy between the present finding
of 20 of 27 WAS patients with WASP-positive PBMC, but only 1 of 13
patients with WASP-positive PBMC in an earlier study (34).
Some of the difference appears due to patient selection, which was
almost random in the present study, whereas the earlier study involved
primarily severe phenotype patients. On the other hand, the
WASP-negative patients in the earlier study included two with the
Val75Met mutation, characterized here for
patients SM and NM as WASP positive.
The correlation of genotype/phenotype in the WAS, proposed by several investigators (3, 7, 28, 36, 37), remains controversial because of several cases of patients with the same mutation and different clinical outcomes (e.g., Refs. 38, 39). Some disparities are caused by difficulty in assigning clinical scores and other confounding factors, including variable quality of medical care and deterioration of T cell number and function over time, so that clinical score can vary with patient age. Despite these problems, there is fairly general agreement that most patients with missense mutations, at least exons 1 and 2 missense mutations, have mild or mild/moderate disease, and most patients with null mutations and some frameshift and splice site mutations have severe disease (reviewed in Ref. 30).
The present study greatly enlarges the primary cell WASP database and demonstrates the absence of WASP in all patient platelets examined, providing a putative molecular explanation for the uniformly severe platelet defect in this disease. The study also identifies unexpected cell lineage discordancy in the processing of some mutated WASP genes and a need to separately analyze T cells and B cells to assess the mutational burden acting on individual patients’ immune cells.
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Acknowledgments |
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Footnotes |
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2 Current address: The Research Institute for Pediatric Hematology and Moscow Medical University, Moscow, Russia.
3 Address correspondence and reprint requests to Dr. Eileen Remold-O’Donnell, Center for Blood Research, 800 Huntington Avenue, Boston, MA 02115. E-mail address:
4 Abbreviations used in this paper: WAS, Wiskott-Aldrich syndrome; WASP, WAS protein; PRP, platelet-rich plasma; ACD, acid-citrate-dextrose.
5 Others reported that normal neutrophils do not express WASP (Ref. 22 ); we cannot explain the discrepant findings.
Accepted for publication September 17, 1999.
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2 assays of 
