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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 2 (January 15), 1998:
pp. 595-602
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
By
Takeshi Futatani,
Toshio Miyawaki,
Satoshi Tsukada,
Shoji Hashimoto,
Toshio Kunikata,
Shigeyuki Arai,
Masashi Kurimoto,
Yo Niida,
Hiroshi Matsuoka,
Yukio Sakiyama,
Tsutomu Iwata,
Shigeru Tsuchiya,
Osamu Tatsuzawa,
Kazuyuki Yoshizaki, and
Tadamitsu Kishimoto
From the Department of Pediatrics, Faculty of Medicine, Toyama
Medical and Pharmaceutical University, Toyama, Japan; the Department of
Medicine III, Osaka University Medical School, Osaka, Japan; the
Fujisaki Institute, Hayashibara Biochemical Laboratory Inc, Okayama,
Japan; the Department of Pediatrics, School of Medicine, Kanazawa
University, Kanazawa, Japan; the Department of Pediatrics, School of
Medicine, Nagoya University, Nagoya, Japan; the Department of
Pediatrics, School of Medicine, Hokkaido University, Sapporo, Japan;
the Department of Pediatrics, Faculty of Medicine, University of Tokyo,
Tokyo, Japan; the Department of Pediatric Oncology, Institute of Aging,
Development, and Cancer, Tohoku University, Sendai, Japan; the Division
of Infectious Disease, National Children's Hospital, Tokyo, Japan; and
the Department of Medical Science I, School of Health and Sport
Sciences, Osaka University, Osaka, Japan.
 |
ABSTRACT |
The B-cell defect in X-linked agammaglobulinemia (XLA) is caused by
mutations in the gene for Bruton's tyrosine kinase (BTK). Using the
anti-BTK monoclonal antibody (48-2H), a flow cytometric analysis of
intracytoplasmic BTK protein expressed in monocytes was successfully
performed. To examine the possible identification of XLA patients and
female carriers by this assay, we studied 41 unrelated XLA families
with (35) or without (6) known BTK mutations. A flow cytometric
assay showed deficient expression of the BTK protein in 40 of 41 patients, complete BTK deficiency in 35, and partial BTK deficiency in
5. One patient exhibited a normal level of BTK expression. All 6 patients with partial BTK deficiency or normal BTK expression had
missense BTK mutations. The cellular mosaicism of BTK
expression in monocytes from obligate carriers was clearly shown in 35 of 41 families. The results suggested that most BTK mutations in XLA
might result in deficient expression of the BTK protein. We conclude
that deficient expression of BTK protein can be evaluated by a flow
cytometric assay, and the clinical usefulness and limitations in
diagnosis of XLA patients and carriers are discussed.
| |
INTRODUCTION |
X-LINKED agammaglobulinemia (XLA) is the
prototypical humoral immunodeficiency first described by Bruton in
1952.1 It is characterized by a paucity of circulating B
cells and a marked reduction in serum levels of all Ig isotypes, which
causes susceptibility to recurrent and severe bacterial infections in
affected males.2-4 The defect in XLA is considered to be
due to inefficient expansion of pre-B cells into later B-cell stages or
incomplete differentiation of B-cell precursors to pre-B
cells.4,5 Using linkage analysis, the XLA gene was first
mapped to the long arm of the human X-chromosome in the region of
Xq21.3-Xq22.6 In early 1993, the gene responsible for XLA
was identified as a cytoplasmic tyrosine kinase, named Bruton's
tyrosine kinase (BTK).7,8 BTK belongs to a group of related
cytoplasmic tyrosine kinases, known as the Btk/Tec family, and consists
of five distinct structural domains, which encompass the N-terminus,
pleckstrin homology (PH) domain, Tec homology (TH) domain, Src homology
3 (SH3) domain, SH2 domain, and the catalytic kinase (SH1)
domain.9-14 The BTK gene analysis has facilitated
the identification of various mutations, including point mutations,
insertions, or deletions, in XLA cases.15 Mutations have
been identified in all five domains of BTK and have been observed to be
associated with a reduction in BTK mRNA, BTK protein, and kinase
activity.8,16-20
In female XLA carriers, B cells manifest the skewed inactivation of the
mutated X-chromosome, reflecting the role of the XLA gene in early
B-cell development.21-23 On the other hand, non-B hematopoietic cells in XLA carriers undergo random inactivation of the
normal and mutated X-chromosomes. The product of the BTK gene
has been detected in B cells and other hematopoietic cells, such as
myeloid cells.8,24,25 Thus, it is possible that
demonstration of BTK mosaicism in non-B hematopoietic cells could lead
to the detection of obligate XLA carriers. In addition, BTK expression in non-B cells must be as clinically informative for evaluation of the
BTK deficiency as the results of BTK mutations in XLA patients, because patients lack circulating B cells.2-4 Generally,
the direct detection of BTK mutations by gene analysis is
time-consuming and labor-intensive in diagnostic studies or the genetic
counseling of XLA families. Furthermore, mutations in the coding
regions of BTK gene are not identified in some cases, even if
they fulfill the criteria for XLA and show no BTK kinase
activity.18
Consequently, we attempted to devise a flow cytometric method for
evaluation of cellular BTK expression using a monoclonal antibody
(MoAb) specific for BTK.18 We show that flow cytometric evaluation of BTK expression in monocytes might constitute a rapid and
sensitive approach for detection of XLA patients and female carriers.
The clinical usefulness of this approach was then assessed in unrelated
41 XLA families.
| |
MATERIALS AND METHODS |
Subjects.
We studied 41 unrelated XLA patients (mean age, 17 years; range, 2 to
34 years) and their mothers in Japan. Thirty-five of these patients had
been analyzed for BTK mutations in previous studies.18,26,27 Among them, 31 patients were found to have mutations in their BTK genes, such as point mutations (missense or
nonsense), deletions, and insertions. In 4 XLA patients (families 32 through 35), no mutations were identified in the coding region of BTK,
although they exhibited markedly reduced levels of the BTK
transcripts.18 Six patients (families 36 through 41) who had not received the BTK genetic analysis were sporadic cases suggestive of XLA, because they exhibited an absence of circulating B
cells and reduced serum Ig levels beginning in early childhood. Healthy
adult volunteers served as normal controls. Heparinized blood samples
of 5 to 10 mL were collected into the syringes containing heparin after
informed consent was obtained.
Cell preparation.
Heparinized venous blood was separated into neutrophils and mononuclear
cells by dextran sedimentation and Ficoll-Hypaque gradient
centrifugation as described.28 Neutrophils were more than
98% pure as assessed by May-Grünwald-Giemsa staining. For immunoblot analysis of the BTK protein, CD3+ T cells,
CD20+ B cells, CD16+ natural killer (NK) cells,
and CD14+ monocytes were purified from mononuclear cells by
an Epics Elite flow cytometer (Coulter Electronics, Inc, Hialeah, FL)
using fluorescein isothiocyanate (FITC)-conjugated corresponding MoAbs,
all of which were purchased from Dako Japan (Kyoto, Japan). The
purified cell populations were more than 95% positive for each marker
as determined by a flow cytometric analysis.
Immunoblot analysis of the BTK protein.
Immunoblot analysis was performed as described
previously.29 In brief, 1 million cells were lysed with 10 µL of lysis buffer (1% Triton-X 100, 10 mmol/L Tris-HCl, pH 7.6, 150 mmol/L NaCl, 5 mmol/L EDTA, 2 mmol/L phenylmethylsulfonyl fluoride, 20 mmol/L  -amino-n-capronic acid, 20 mmol/L iodoacetamide, 0.01%
soybean trypsin inhibitor, and 10 µg/mL aprotinin) for 40 minutes on
ice and were centrifuged for 10 minutes at 15,000g. The
supernatants were mixed with an equal volume of sodium dodecyl sulfate
(SDS) sample buffer and boiled for 3 minutes. The samples were size fractionated on a 10% SDS-polyacrylamide gel and electroblotted on
nitrocellulose filters. The blots were blocked in 5% skim milk in
phosphate-buffered saline (PBS; pH 7.4) for 1 hour, reacted with
anti-BTK (48-2H: IgG1) MoAb18 at 2 µg/mL or
anti- -actin MoAb (Sigma Chemical Co, St Louis, MO) at 1 µg/mL for
1 hour, and then incubated with a 1:2,000 dilution of
peroxidase-labeled goat antimouse IgG antibody (Biosource
International, Camarillo, CA) for 1 hour. The blots were rinsed at
least four times for 10 minutes in PBS with 0.05% Tween 20 between
incubations. Blots were developed by using the ECL Western blotting
detection system (Amersham International plc, Amersham, UK). Prestained
molecular weight markers (Rainbow colored protein; Amersham
International plc) were used as the molecular size standard.
Flow cytometric assay.
Intracellular staining with anti-BTK MoAb was performed as
described.29 The cells were first fixed in 4%
paraformaldehyde in PBS for 20 minutes at room temperature and then
permeabilized in 0.1% Triton X-100 in Tris-buffered saline (pH 7.4)
with 1 mg/mL bovine serum albumin for 5 minutes. Subsequently, these
fixed, permeabilized cells were reacted with 2 µg/mL of anti-BTK
(48-2H) or control IgG1 (Dako Japan) MoAbs for 20 minutes on ice,
washed, and then incubated with a 1:2,000 dilution FITC-conjugated goat antimouse IgG1 antibody (Zymed Laboratories, San Francisco, CA) for 20 minutes. In some experiments, mononuclear cells were stained with
phycoerythrin (PE)-labeled CD20 (IgG2a; Coulter Immunology, Hialeah,
FL) or CD14 (IgG2a; Dako Japan) MoAb before cellular permeabilization
to discriminate B cells or monocytes, respectively, from other cells.
The stained cells were analyzed on a Cytoron Absolute flow cytometer
(Ortho-Clinical Diagnostics, Tokyo, Japan).
| |
RESULTS |
We first performed an immunoblot analysis of T cells, B cells, NK
cells, monocytes, and neutrophils using the anti-BTK MoAb (48-2H).
Figure 1 shows representative results
obtained using cells from a normal adult donor. A 77-kD band identical
to the BTK protein was detected in the lysate of B cells, but not of T
cells, NK cells, or neutrophils. Importantly, the same band was evident
in monocytes. It should be stressed that the anti-BTK MoAb detected
very few additional bands in the immunoblot. We further examined
whether a flow cytometric assay using this antibody could show
different BTK expression among blood leukocyte populations. The cells
were fixed and permeabilized to detect intracytoplasmic BTK protein. As
shown in Fig 2A, two-color analysis of
lymphocytes showed that the BTK protein was expressed in most B cells,
but not in non-B cells. Furthermore, monocytes, but not neutrophils, expressed BTK (Fig 2B).
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| Fig 1.
Immunoblot detection of BTK expressed in blood leukocyte
populations from normal donors. Neutrophils were isolated from the blood by dextran sedimentation and Ficoll-Hypaque gradient
centrifugation. CD3+ T cells, CD20+ B
cells, CD16+ NK cells, and CD14+ monocytes
were purified from mononuclear cells by electronic sorting. The cells
were subjected to immunoblot analysis using anti-BTK or anti--actin
MoAbs.
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| Fig 2.
Flow cytometric analysis of BTK expression in blood
leukocytes from normal donors. (A) Two-color immunofluorescence
analysis of BTK expression in B cells and non-B cells. Mononuclear
cells were stained with PE-labeled anti-CD20 MoAb, fixed, and
permeabilized. The cells were then reacted with anti-BTK or irrelevant
control MoAbs and further incubated with FITC-labeled secondary
antibody. The dot plot map (a) shows the two-color pattern of
lymphocytes gated by forward and right angle right scatter. A second
gate was set to include CD20+ B cells (b) and non-B cells
(c), and the BTK expression in each cell population is presented as a
histogram. (B) Analysis of BTK expression in monocytes and neutrophils.
Mononuclear cells or neutrophils were stained for BTK as described
above. BTK expression in monocytes was evaluated by gating on
CD14+ population. The dashed line indicates the control
antibody. Five thousand cells were evaluated in each gated
population.
|
|
We then used the flow cytometric assay to evaluate BTK expression in
XLA patients and to detect cellular mosaicism of BTK expression in
their mothers as obligate carriers. Figure
3 shows one example from family 19 with deletion in the PH domain.
Whereas B cells were markedly decreased in the blood of the patient,
the mother possessed a substantial number of circulating B cells
expressing BTK (Fig 3A). As shown in Fig 3B, the patient obviously
exhibited negligible expression of the BTK protein in monocytes. In
addition, the flow cytometric assay demonstrated the clearly bimodal or mosaic pattern of BTK expression in monocytes of the mother, indicating that they consisted of both BTK+ and BTK
cells.
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| Fig 3.
Flow cytometric analysis of cellular BTK expression in a
normal donor and in an XLA patient (family 19) and his mother. Dot plot
maps of BTK versus CD20 gated for lymphocytes (A) and histograms gated
for CD14+ monocytes (B) were obtained as described in the
legend of Fig 2. Five thousand cells were evaluated in each gated
population. The dashed line indicates the control antibody.
|
|
To further validate the flow cytometric assay, we analyzed 41 unrelated
XLA patients and their mothers. The results, including known BTK
mutations, are summarized in Table 1.
Figure 4 shows the relationships between
patients and mothers as to monocyte BTK expression profiles. In most of
the patients (40 of 41 families) studied, the flow cytometric assay did
not detect significant amounts of BTK protein or only detected markedly
reduced levels of BTK protein, despite the intense BTK expression in 32 normal controls. This BTK deficiency status was almost complete (<1% anti-BTK stainable or positive cells in monocytes) in 35 patients (Fig
4A and B). In 5 (families 1 through 4 and 7) patients showing BTK
deficiency, the low but detectable levels of BTK (4% to 32% positive)
seemed to be expressed in monocytes (Fig 4C). Arbitrarily, we called
the former and the latter complete and partial BTK deficiencies, respectively. The complete BTK deficiency included various BTK mutations such as nonsense, insertion, deletion, and even missense. The
complete BTK deficiency was also found in 4 patients (families 32 through 35) who had no mutations in the BTK coding region but exhibited
reduced levels of BTK transcripts.18 Six sporadic cases (families 36 through 41) who had not received genetic analysis showed the complete BTK deficiency as well. The partial BTK deficiency was only detected in cases with missense mutations (families 1 through
4 and 7), which might reflect the partially impaired stability of the
BTK protein. As shown in Fig 4D, the patient in family 6, who had the
missense mutation in the catalytic loop of the SH1
domain,18 exhibited the intense expression of the BTK
protein in monocytes (97% positive) comparable to that of normals.
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| Fig 4.
Relationship of monocyte BTK expression profiles between
XLA patients and their mothers. Each histogram represents analysis of
CD14+ monocytes. The solid and dashed lines indicate the
staining with anti-BTK and control antibodies, respectively. Five
thousand cells were evaluated in each gated population. (A) The patient
had the complete BTK deficiency, and the mother showed the mosaic BTK expression (eg, family 13). (B) The patient had the complete BTK deficiency, but the mother showed the single-positive BTK expression (eg, family 14). (C) The patient had the partial BTK deficiency, and
the mother showed the mosaic BTK expression with dimly
BTK+ cells and brightly BTK-expressing cells (eg, family
7). (D) Both the patient and the mother expressed the normal BTK
expression (eg, family 6). The frequency (no. of families/total no. of
families) is depicted below the histogram.
|
|
The patterns of BTK expression in mothers' monocytes are summarized in
Table 1 and Fig 4. The bimodal pattern of BTK expression in monocytes
may indicate cellular mosaicism in mothers as obligate XLA carriers.
However, the ratio of brightly staining BTK+ monocytes to
weakly staining BTK+ monocytes varied from mother to mother
(Table 1). The flow cytometric assay disclosed that normal monocytes
frequently had approximately 3% BTK cells. We
classified mothers' monocytes as mosaic when the percentage of
brightly anti-BTK-stainable monocytes was less than 90%, which was
almost equal to the mean minus 3 SD of positivity of monocyte BTK
expression in normals. Based on this criterion, the mosaic BTK patterns
in the mothers' monocytes were obtainable in 35 of 41 (~85%) of
families (Table 1 and Fig 4). This mosaicism was observed in the
majority of families with complete or partial BTK deficiencies (Fig 4A
and C). The mosaicism in the mother's monocytes was also found in all
families without any identifiable mutations (families 32 through 35)
and 5 sporadic cases (families 36 through 40), supporting the
BTK-related agammaglobulinemia in both groups. In the remaining 6 families (6, 14, 15, 17, 23, and 41), the mosaic pattern of BTK
expression in the mothers' monocytes was not obtained (Fig 4B and D).
The normal pattern of monocyte BTK expression was expected in the
mother of family 6, because the patient expressed BTK at the normal
levels, which might lead to the nonmosaic BTK expression in the
mother's monocytes even in the presence of X-chromosomal mosaicism
(Fig 4D). In the other 5 families (14, 15, 17, 23, and 41), all of
which are sporadic cases, the patients showed the complete BTK
deficiency. The single positive pattern of BTK expression in mothers'
monocytes in these cases raised the possibility that BTK deficiency in
the patients might arise from the de novo mutation or gonadal
mosaicism.30 Nucleotide sequence analysis of the genomic
BTK gene in the white blood cells of mothers was performed for
families 14 and 15. The analysis showed that the mutated allele was
absent in somatic cells of both mothers, supporting the possibility
discussed above (data not shown). It is also possible that the
excessively skewed inactivation of the mutated X-chromosome may occur
in the mothers' monocytes.31 However, these two
possibilities were not examined for the mothers in families 17, 23, and
41.
| |
DISCUSSION |
We have described a practical flow cytometric assay for identifying
affected males and female carriers in XLA families. The immunoblot
analysis using the anti-BTK MoAb (48-2H) showed that the BTK protein
was selectively expressed in monocytes and B cells among blood
leukocytes. Other investigators have also observed BTK expression in
monocytes as well as B cells.19 Although several laboratories have generated MoAbs or antisera against the BTK protein,8,25,32,33 these antibodies have been shown to
cross-react with proteins other than BTK in the immunoblot analysis or
immunoprecipitation. In contrast, our anti-BTK MoAb 48-2H detects the
BTK protein with very few additional bands in the immunoblot, implying
the suitability of this antibody for flow cytometric use. Consistent
with the results of the immunoblot analysis, we showed that a flow
cytometric assay could identify preferential expression of the
intracytoplasmic BTK protein in both monocytes and B cells.
A flow cytometric assay on monocyte BTK expression was used to evaluate
BTK deficiency status and to compare with BTK mutations in XLA
patients. This assay showed that, in almost all of the XLA patients
(~98%) studied here, BTK protein was absent or weakly detectable in
monocytes, whereas strong BTK expression was found in monocytes from
normal donors. Our anti-BTK MoAb was generated against the SH3 domain
of BTK.18 It was predictable that the defective BTK protein
expression was demonstrated in patients with mutations causing the
premature termination at the 3 terminal region from the SH3
domain (families 8 through 10, 17 through 21, and 31) or the deletion
of the SH3 domain-containing region (families 22 and 23). Additionally,
it was predicted that 4 patients (families 32 through 35) in whom
reduced levels of BTK mRNA had been found despite the absence of
mutations in the coding region18 might exhibit defective
BTK expression. However, these cases were only 15 (37%) of 41 families
examined. In nonsense mutations (families 11 through 16), deletions
(families 24 through 28), or insertions (families 29 and 30) not
affecting the SH3 domain, the flow cytometric assay would likely detect
BTK proteins with various sizes. Nevertheless, the defective BTK
expression was observed in all of these cases in our assay.
Additionally, in the cases with several missense mutations, the mutated
BTK proteins were detected at markedly reduced levels, which is
consistent with our previous observation that some of the BTK proteins
carrying missense mutations in the SH1 domains were not detected by
anti-BTK immunoblotting.18 The normal BTK expression
pattern, suggesting the production of normal amounts of altered BTK,
was found only in 1 case (family 6) with a missense mutation in the
catalytic loop of the SH1 domain.18 Vihinen et
al34 have described a similar case with the missense mutation in the same loop.
The majority of the nonsense mutations and deletions may unstabilize
the mRNA or the protein, as has been shown in previous studies on
BTK8,16-20 and other genes.35,36
Furthermore, it has been reported that several missense mutations
result in the reduced stability of the BTK protein16,18 and
other proteins such as WASP,37 especially in hematopoietic
cells. However, it must be noted here that failure to detect mutated
BTK proteins does not necessarily imply the absence or instability of
proteins. There remains an alternative possibility that the anti-BTK
MoAb (48-2H) used in this study might be unable to recognize some of
the mutated BTK proteins. Although our unpublished data indicated that
this antibody can recognize several artificially mutated BTK proteins
(including Arg28 to Pro, Gln41 to Lys, Trp124 to Gly, Tyr223 to Phe,
Trp251 to Leu, Lys430 to Arg, Tyr551 to Phe mutations, and, in
addition, a truncated protein only having the unique region and SH3
domain), it is impossible to test the latter possibility for all
mutated BTK proteins studied here, and further studies using other
anti-BTK antibodies will be necessary. Collectively, the present study in 41 unrelated Japanese XLA families showed that most (40 of 41)
mutations in XLA, including 5 different missense mutations, resulted in
the absence or reduced levels of the BTK protein detectable by a flow
cytometric assay using the MoAb 48-2H. A database of BTK mutations has
currently compiled 318 unrelated XLA families, which includes 123 families carrying missense mutations.15 To understand the
precise molecular mechanism of the pathogenesis of XLA and
genotype-phenotype relationships, it may be important to investigate
which mutations result in the instability of BTK proteins or which
mutations result in the loss of the function of some domain without
affecting the stability of BTK proteins. The flow cytometric analysis
with combinations of MoAb described here and other antibodies will be
useful in this regard.
It is noteworthy that the Arg28 mutation to Pro mutation (family 1),
which is located in the amino acid residue in the mutation of XID
mice,38 resulted in a greatly reduced level of BTK protein expression. In our previous report,18 we showed a similar
level of kinase activity in the Arg28 to Pro-mutated BTK, comparable to
that of the wild-type BTK. This discrepancy is apparently due to the
difference in experiment methodologies. The in vitro kinase assay and
the immunoblot assay described in our previous report18 were performed under nonquantitative conditions because of the weak
kinase activity detected in peripheral blood cells and the limited
availability of clinical samples. Our data in the present study
indicate that the flow cytometric analysis is much more quantitative in
the detection of BTK expression compared with other methods described
in our previous report.18 In the PH domain of BTK, the
Arg28 residue has been postulated to be located within a binding site
of potential ligands such as phospholipids39 or protein
kinase C,40 and in vitro experiments using GST fusion proteins have suggested that the Arg28 mutation diminishes binding capabilities of these potential ligands.39,40 Our
observation that the Arg28 (to Pro) mutation reduced the expression of
BTK protein measured by the flow cytometric analysis might suggest a
new interpretation on the significance of the Arg28 mutation. Consistent with our observation, other investigators observed a
decreased expression of the BTK protein harboring the Arg28 to Cys
mutation (XID mutation) in murine mast cells (Hata et al, personal
communication, October 1997). The Arg28 mutation may lead
to a conformational change of BTK that makes the protein unstable, at
least in some intracellular conditions within hematopoietic cells.
Alternatively, this mutation may disrupt the binding of potential
ligands that are necessary for the stabilization of the BTK protein.
We showed that a flow cytometric assay based on monocyte BTK expression
is useful to identify the X-chromosomal mosaicism in mothers as
obligate carriers. In 35 of 41 families (~85%), it was found that
the mothers exhibited a bimodal or mosaic BTK expression in monocytes.
During this study, we had the opportunity to examine 14 sisters of 11 patients with BTK deficiency. The flow cytometric assay showed that, in
9 of 14 sisters (~64%), bimodal expression of BTK in monocytes was
detected, implying XLA carriers. Unexpectedly, an apparently normal
profile of monocyte BTK expression in mothers was obtained in 6 families (6, 14, 15, 17, 23, and 41). This was expected in family 6, because the patient showed strong monocyte BTK expression comparable to
that of normal patients. Patients in the other 5 families showed the
complete BTK deficiency. All of these patients are sporadic cases
without any family history. The nonmosaic expression of BTK in their
mother's monocytes may be derived from the de novo mutation in oocytes or gonadal mosaicism that have been seen in certain X-linked hereditary diseases.30 Such a kind of inheritance was confirmed in
families 14 and 15 by nucleotide sequence analysis of the mother's
somatic cells. It has been observed that X-chromosome inactivation may result in skewing in normal cells.31 It is possible that
the skewed inactivation of the mutated X-chromosome may result in the
single positive pattern of BTK expression in the mother's monocytes.
In the course of this study, we determined whether the skewed
inactivation of the mutated X-chromosome might occur in monocytes from
female XLA carriers. In family 29, although the mother showed mosaic
BTK expression in monocytes, the single-positive pattern of monocyte
BTK expression was found in the grandmother. Using the genomic
analysis, we found that both normal and mutated X-chromosomes were
present in somatic cells of the grandmother as well as the mother
(unpublished observations, October 1997). Thus, it should
be noted that a normal pattern of monocyte BTK expression in the woman
does not rule out the possibility that she is an XLA carrier. Of
course, the clinical usefulness of a flow cytometric assay for
identification of XLA carriers should be validated by further analysis
of additional cases in a combination with the genetic analysis.
In conclusion, the present study suggests that most BTK
mutations appear to result in deficient expression of the BTK protein in affected individuals. The mosaic expression of the BTK protein in
monocytes, as shown in a flow cytometric assay, is potentially useful
in the diagnosis of female carriers. With an accumulation of
BTK mutations, atypical or mild XLA has been found among the cases previously diagnosed as common variable immunodeficiency or other
immunodeficiencies,4,11,16,18 and the clinically different
phenotypes have been observed in siblings with the same BTK
mutations.19 The semiquantitative capability of a flow
cytometric assay to assess the cellular BTK expression may contribute
to an elucidation of the significance of BTK mutations in
phenotypic expression of the disease and an understanding of the role
of BTK in B-cell development.
| |
FOOTNOTES |
Submitted May 27, 1997;
accepted September 19, 1997.
Supported by Grant-in-Aid for Scientific Research from the Ministry of
Education, Science and Culture of Japan and by grants from the Ministry
of Health and Welfare of Japan and from the Mother and Child Health
Foundation.
Address reprint requests to Toshio Miyawaki, MD, PhD, Department of
Pediatrics, Faculty of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama, Toyama 930-01, Japan.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors are very grateful to all families and physicians for the
generous cooperation in this study and to Dr Toshiaki Kawakami and his
colleagues (La Jolla Institute for Allergy and Immunology, San Diego,
CA) for the kind supply of their preliminary data and helpful
discussion. We also thank Hitoshi Moriuchi for the excellent technical
assistance.
| |
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Abstract
Introduction
Abstract