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Keywords:

  • X-linked agammaglobulinaemia;
  • Bruton's tyrosine kinase;
  • flow cytometry;
  • platelet;
  • carrier detection

Abstract

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

X-linked agammaglobulinaemia (XLA) is a primary immunodeficiency caused by mutations in the gene coding for Bruton's tyrosine kinase (Btk) and is characterized by an arrest of B-cell development. We analysed Btk protein expression in platelets using flow cytometry and found that normal platelets express large amounts of Btk. Assessment of affected males from 45 unrelated XLA families revealed that platelets of the majority of the patients (37 out of 45 families) had decreased or absent Btk expression, and that platelets from carrier females of these families had both normal and mutated Btk expression, indicating that megakaryocytes in XLA carriers undergo random X-chromosome inactivation. These observations demonstrate that Btk is not crucial for maturation of megakaryocytes and the production of platelets. No correlation between Btk expression in platelets and clinical phenotype was observed in this study. Flow cytometric evaluation using platelets is a simple and rapid method to test Btk expression. It may be used as a screening test for XLA and for carrier detection, followed, if necessary, by more expensive mutation analyses.

X-linked agammaglobulinaemia (XLA), first described by Bruton (1952), remains the prototypic example of a primary immunodeficiency disease in which the defect is limited to B lymphocytes. As a result, affected males have profoundly abnormal antibody production and present with recurrent bacterial infections (Ochs & Smith, 1996). XLA is caused by mutations of Bruton's tyrosine kinase (Btk) which is required for B-cell development (Vetrie et al, 1993; Tsukada et al, 1993). Btk is present in pre-B cells and B lymphocytes, but not in plasma cells and T lymphocytes. Btk is a member of the cytoplasmic Tec family of tyrosine kinases and consists of a pleckstrin homology (PH) domain, a Tec homology (TH) domain, a Src homology 3 (SH3) domain, a SH2 domain and a SH1 (kinase) domain. Mutations of Btk, which include missense and nonsense mutations, deletions, insertions and splice site mutations (Vetrie et al, 1993), are distributed throughout the gene and may result in complete absence of the protein, or the presence of truncated or full-length non-functional proteins.

The clinical spectrum of XLA varies, not only from family to family (Ochs & Smith, 1996), but also within families with more than one affected member (Wedgwood & Ochs, 1980; Kornfeld et al, 1997).

Because of this variability in the clinical phenotype, the diagnosis of XLA may be difficult in some cases (Ochs & Smith, 1996). The most decisive approach for confirming the diagnosis of XLA is mutation analysis of Btk. Once the mutation in a given family has been determined, carrier females from that family can be readily identified. However, sequence analysis of the Btk gene requires a specialized laboratory, is time consuming and may be difficult. A simple screening test that allows easy identification of XLA patients and carrier females has recently been designed, based on the fact that monocytes express Btk and, in contrast to B cells, undergo random X-chromosome inactivation (Futatani et al, 1998). To further simplify the screening procedure, we have studied Btk in platelets and found that normal platelets have large amounts of Btk, that platelets of the majority of XLA patients have decreased or absent Btk and that platelets of carrier females have both normal and mutated Btk, suggesting that X-chromosome inactivation is random in megakaryocytes.

Patients and methods

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Patients In this study we included 55 patients from 45 unrelated XLA families with known mutations of Btk (Table I), their carrier mothers and other female relatives at risk. The majority of patients presented with classic clinical and laboratory findings of XLA including recurrent bacterial infections, low serum immunoglobulin levels and less than 1% peripheral blood B cells. Several patients, however, had a milder form with late onset and few infections.

Table I.   Btk expression in platelets from 55 XLA patients from 45 related families with known Btk mutation.
Family/BtkCodonAffectedBtk expression pattern*
patientmutationchangedomainPatientsCarriers
  • *

    Btk expression pattern according to the staining intensity: I, no expression or very reduced expression of Btk; II, reduced but substantial expression of Btk. Clearly distinguished from a normal control; III, normal or slightly reduced expression of Btk.

  • Single peak in carrier females owing to normal expression level of mutated Btk protein.

  • MSP, multiple splice products; ND, analysis not done.

  • Exon 14,15,16 skipped and other aberrant splice products.

  • §

    Exon 15,16 skipped, other aberrant splice products and normal product.

  • Exon17 skipped, exon 15,16,17 skipped and other aberrant splice products.

  • **

    Exon17 skipped, other aberrant splice products and normal product.

  • ††Nucleotide position according to Vetrie et al (1993).

Missense mutations (n = 22 families/27 patients)
B-08-1/2G215AArg 28 HisPHIIINormal
B-10-1G215AArg 28 HisPHIIINormal
B-11-1G215AArg 28 HisPHIIINormal
B-09-1/2G215AArg 28 HisPHIIINormal
B-12-1A229CThr 33 ProPHIMosaic
B-23-1G593CCys154 SerTHIIMosaic
B-34-1G995AArg 288 GlnSH2IIINormal
B-86-1/2G995AArg 288 GlnSH2IIIND
B-36-1A1223CHis 364 ProSH2IIMosaic
B-42-1T1355CLeu 408 ProSH1IIMosaic
B-43-1T1384CTyr 418 HisSH1IIND
B-46-1A1493GHis 454 ArgSH1IIMosaic
B-48-1T1589CLeu 486 ProSH1IMosaic
B-55-1G1691AArg 520 GlnSH1IIINormal
B-56-1G1691AArg 520 GlnSH1IIINormal
B-61-1G1706AArg 525 GlnSH1IIMosaic
B-62-1/2/3/4G1706AArg 525 GlnSH1IIMosaic
B-64-1G1712TCys 527 PheSH1IMosaic
B-75-1/2A1898GGlu 589 GlySH1IMosaic
B-77-1G1970AGly 613 AspSH1IIMosaic
B-82-1T2072CLeu 647 ProSH1IIMosaic
B-84-1T2075CLeu 648 ProSH1IND
Nonsense mutations (n = 7 families/8 patients)
B-17-1/2C403TGlu 91 stopPHIMosaic
B-21-1T473G & C474APhe114 stopPHIMosaic
B-24-1C832TGln 234 stopSH3IMosaic
B-25-1G805TGlu 240 stopSH3IIMosaic
B-26-1G885ATrp 251 stopSH3IMosaic
B-54-1C1690TArg 520 stopSH1IND
B-76-1A1930TArg 600 stopSH1INormal (new mutation)
Genomic insertions, deletions (n = 8 families/9 patients)
B-03-1del 26 bp (146–171)FS[RIGHTWARDS ARROW]stopPHIND
B-07-1del C (173–175)FS[RIGHTWARDS ARROW]stopPHIND
B-13-1ins A (258–259)FS[RIGHTWARDS ARROW]stopPHIMosaic
B-16-1/2del AG (330–331)FS[RIGHTWARDS ARROW]stopPHIMosaic
B-22-1ins 47bp (505–506)FS[RIGHTWARDS ARROW]stopPHIMosaic
B-40-1ins 6 bp (1263–1264)FS[RIGHTWARDS ARROW]stopSH1IMosaic
B-41-1ins G (1297–1298)FS[RIGHTWARDS ARROW]stopSH1IMosaic
B-72-1del G (1830)FS[RIGHTWARDS ARROW]stopSH1INormal (skewed)
Splice site mutations (n = 7 families/10 patients)
B-18-1intron 4 (−2)a[RIGHTWARDS ARROW]c21 bp insPHIND
B-30-1intron 9 (+1)g[RIGHTWARDS ARROW]aexon 9 skipSH3IMosaic
B-44-1intron 14 (+5)g[RIGHTWARDS ARROW]aMSPSH1IIMosaic
B-59-1/2/3intron15 (−9 to −12), 4 bp delMSP§SH1IMosaic
B-67-1intron16 (−3)c[RIGHTWARDS ARROW]aMSPSH1IMosaic
B-68-1intron 16 (−2)a[RIGHTWARDS ARROW]cMSP**SH1IIMosaic
B-74-1/2G1882Texon 17 skipSH1IMosaic
Others (n = 1)
B-01-1−67a[RIGHTWARDS ARROW]c(promoter)††normalpromoterIIMosaic

Platelet preparation Blood was collected by venepuncture using siliconized vacutainer tubes containing 1/10 volume of 3·8% trisodium citrate. Platelet-rich plasma (PRP) was prepared by centrifugation at 120 g for 15 min at room temperature. Aspirin (Sigma Chemical, St Louis, MO, USA) was added at a concentration of 2 μmol/l and then incubated for 30 min at room temperature. Platelets were sedimented by centrifugation at 1200 g for 20 min and resuspended in a modified Tyrode's buffer [137 mmol/l NaCl, 2 mmol/l KCl, 12 mmol/l NaHCO3, 0·3 mmol/l NaH2PO4, 5·5 mmol/l dextrose, 5 mmol/l HEPES, 5 mmol/l EDTA and 0·35% bovine serum albumin (BSA), pH 7·4].

Mononuclear cell preparation Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized venous blood using Ficoll-Hypaque gradient centrifugation. For immunoblot analysis of the Btk protein, CD3+ T cells, CD20+ B cells and CD3+CD14+ monocytes were purified from PBMCs by cell sorting using a FACStar (Becton and Dickinson).

Flow cytometric analysis Platelets (1 × 108) were fixed with an equal volume of 4% paraformaldehyde in phosphate-buffered saline (PBS; pH 7·4) for 15 min. Fixed platelets were washed twice and permeabilized with 0·1% Saponin in modified Tyrode's buffer for 20 min. Intracellular staining was performed with anti-Btk monoclonal antibody (mAb) 48–2H (IgG1), which recognizes an epitope within the SH3 domain, at a final concentration of 20 μg/ml or with the isotype control (Southern Biotechnology Associates, Birmingham, AL, USA) for 20 min. After washing twice, samples were incubated with a 1:100-diluted fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG1 antibody (Southern Biotechnology Associates) for 20 min. Samples were washed twice and analysed using flow cytometry (FACScan; Becton and Dickinson) All procedures were performed at room temperature.

PBMCs were stained with anti-Btk mAbs as described (Futatani et al, 1998). Briefly, PBMCs were first stained with phycoerythrin (PE)-conjugated anti-CD14 (IgG2a, DAKO, Carpinteria, CA, USA) for identification of monocytes, anti-CD20 (IgG2b; Ancell, Bayport, MI, USA) for B cells, and anti-CD4 and anti-CD8 (IgG2b and IgG2a; Ancell) for T cells. The cells were fixed with 4% paraformaldehyde in PBS, and then permeabilized with 0·1% Triton X-100 in TBS (pH 7·4) containing 0·1% BSA for 5 min at room temperature. Fixed, permeabilized cells were reacted with 2 μg/ml of anti-Btk mAb 48–2H or with isotype control for 20 min on ice. After washing twice, cells were incubated with a 1:1000 dilution of FITC-conjugated anti-mouse IgG1 antibody for 20 min on ice and analysed using flow cytometry.

Immunoblot analysis Purified cells were lysed with lysis buffer (1% Triton-X, 10 mmol/l Tris-HCl pH 7·6, 150 mmol/l NaCl, 5 mmol/l EDTA, 2 mmol/l phenylsulphonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin and 20 mmol/l iodoacetamide) for 40 min on ice and centrifuged. Protein concentrations of the lysates were measured with a Bio-Rad DC protein assay (Bio-Rad, Hercules, CA, USA). A total of 30 μg of lysate was mixed with an equal volume of sodium dodecyl sulphate (SDS) sample buffer and boiled for 5 min. The samples were size-fractionated on a 10% SDS polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon P; Millipore, Bedford, MA, USA). After blocking with 5% skimmed milk in PBS, the membranes were incubated with anti-Btk mAb (48–2H) or anti-β actin polyclonal antibody (Sigma Chemical) and then washed and exposed to a 1:2000 dilution of peroxidase-conjugated goat anti-mouse immunoglobulin (Biosource International, Camarillo, CA, USA). Blots were visualized using the enhanced chemiluminescence (ECL) system (Amersham, Arlington Heights, IL, USA) according to the manufacturer's instructions.

X-chromosome inactivation assay The X-chromosome inactivation status was evaluated by the analysis of DNA methylation at the human androgen receptor gene (HUMARA). Genomic DNA (500 ng) from neutrophils was digested with 10 U of RsaI alone or RsaI with 20 U of HpaII at 37°C overnight in 20 μl of reaction mixture. Primer sequences were as follows: For the forward primer: 5′-GCTGTGAAGGTTGCTGTTCCTCAT-3′; for the reverse primer: 5′-TCCAGAATCTGTTC CAGAGCGTGC-3′. From this reaction mixture, 2 μl was added in a total volume of 25 μl polymerase chain reaction (PCR) mixture containing 200 μmol/l dNTP, 0·2 U of Taq polymerase, 1·5 mmol/l MgCl2, 0·5 μmol/l each primer, 0·05 μmol/l [γ-32P] end-labelled forward primer and 2·5 μl of 10× Taq buffer. Following an initial denaturation step of 5 min at 94°C, 25 amplification cycles were performed, 60 s at 94°C, 30 s at 60°C and 30 s at 72°C. In the final cycle, extension at 72°C was prolonged to 10 min. A 1:1 dilution (2 μl) of the amplification products was then electrophoresed through a non-denaturing 7% acrylamide gel. The gel was dried and exposed to X-ray film at −80°C for 12 h.

Results

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Btk is strongly expressed in platelets

To determine whether platelets express Btk protein, we used immunoblotting to analyse Btk expression by platelets compared with T cells, B cells and monocytes. Immunoblot analysis using mAb 48–2H showed that a 77-kDa band, corresponding to the Btk protein, was strongly expressed in platelets and the expression level was similar to that of B cells and monocytes (Fig 1A).

image

Figure 1.  Btk expression in peripheral blood cells from normal donors. (A) Btk expression in peripheral blood cells using immunoblot analysis. CD3+ T cells, CD20+ B cells and CD14+ monocytes were purified by electronic sorting. Platelets were isolated from platelet-rich plasma. Lysates (30 μg) from each cell population were resolved on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with anti-Btk monoclonal antibody 48–2H. (B) Btk expression in peripheral blood cells using flow cytometry. T cells, B cells and monocytes (but not platelets) were first stained with phycoerythrin (PE)-conjugated anti-CD3, anti-CD20 and anti-CD14 monoclonal antibodies (mAbs) respectively. The cells were fixed and permeabilized and reacted with anti-Btk mAb 48–2H, followed by staining with anti-mouse IgG1 fluorescein isothiocyanate (FITC)-conjugated secondary antibody. Btk-stained cells are shown by the solid lines. Cells stained with isotype-matched control mAbs are shown by the dashed lines.

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To assess intracellular Btk expression by normal platelets, we used flow cytometry after permeabilization and Btk staining. As shown in Fig 1B, more than 95% of normal platelets expressed Btk, consistent with the immunoblot results.

Btk expression is deficient in XLA patients and carrier females

To determine whether the Btk signal observed in immunoblots from normal platelet extracts was specific for Btk, we evaluated Btk expression by platelets from affected members of one family with a nonsense mutation in the PH domain (family B-21) and of another family with a missense mutation in the SH2 domain (family B-36). As shown in Fig 2, platelets from the patient with the nonsense mutation completely lacked Btk expression, whereas those from the patient with the missense mutation expressed Btk, although the density of the band was markedly weaker than that of the normal control. In addition, the amounts of Btk expressed by platelets from the patients' carrier mothers were reduced compared with normal controls. The finding of reduced Btk expression in platelets of carrier females suggests random X-chromosome inactivation in platelet progenitor cells.

image

Figure 2.  Btk expression in platelets from XLA patients and carrier females. Platelet lysates (30 μg) from XLA patients with either a nonsense mutation in the PH domain (patient1) or a missense mutation in the SH2 domain (Patient 2), or from their mothers, were resolved using SDS-PAGE and immunoblotted with anti-Btk mAb 48–2H.

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To evaluate the usefulness of flow cytometry to analyse Btk in platelets as a simple diagnostic tool to screen for XLA, we examined 55 patients from 45 unrelated and unselected XLA families with known mutations of Btk, whose characteristics are detailed in Table I. Mutations of Btk in these families included 22 missense and seven nonsense mutations, eight genomic insertions or deletions, seven splice site mutations and one mutation affecting the promoter region of Btk. Representative flow cytometric patterns of Btk expression are shown in Fig 3. Three groups could be identified according to the intensity of Btk staining. Type I pattern was defined as no or very reduced expression of Btk in platelets, type II pattern as reduced but substantial Btk expression, and type III pattern as normal or minimally reduced expression of Btk. In analogy, carrier females from families with pattern I or II staining of platelets showed two peaks of Btk, representing a population of normal expression and a second population with abnormal Btk expression. This observation demonstrates that platelets of females known to be carriers for XLA are derived from megakaryocytes with random X-chromosome inactivation, suggesting that megakaryocytes with mutated Btk do not have a disadvantage as to maturation and production of platelets compared with normal megakaryocytes. Btk expression by monocytes was assessed in parallel and showed an identical pattern both in XLA patients and carrier females, including the same ratio of normal and abnormal Btk levels in carrier females (data not shown).

image

Figure 3.  Representative Btk expression pattern in platelets assessed using flow cytometry. Permeabilized platelets stained with anti-Btk mAb 48–2H are shown by the solid lines. Platelets stained with isotype-matched control antibody are shown by the dashed lines. The type I pattern is observed in XLA patients with very reduced or no expression of Btk; carrier females of type I families have two population of platelets, one with normal and one with absent expression of Btk. In the type II pattern, Btk expression is reduced and carrier females have two population of cells, one with normal and one with reduced expression of Btk. The type III pattern indicates normal expression of Btk in platelets from patients and carrier females.

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Of a total of 45 families with XLA studied, we observed a type I pattern in 24 families (52%). Every patient with a genomic insertion or deletion resulting in frame shift and premature termination, and four out of five patients with nonsense mutations, showed a type I pattern. A type I pattern was also observed in six out of 22 families with missense mutations.

A type II pattern was observed in 14 XLA families (30%). Of those, nine had missense mutations, one had a nonsense mutation in the SH3 domain and three had splice site mutations. One family with a type II pattern and a mild phenotype (family B-01) was found to have a single nucleotide substitution affecting the PU.1 binding site in the promoter region of Btk. Flow cytometric analysis of both platelets and monocytes from this patient showed a substantial amount of Btk that was of normal sequence. This observation suggests that the mutation affecting the PU.1 binding site does not abrogate the promoter activity completely, either because of some remaining binding of PU.1 or because of the use of other transcriptional factor binding sites such as Sp1.

A type III pattern was observed in eight unrelated families (18%) with three different missense mutations, all involving an arginine (A28, A288, A520). In families with the type III pattern, patient and carrier detection using flow cytometric analysis of platelets or monocytes was not possible because of the normal amount of mutated Btk protein expressed.

A total of 59 potential female carriers were analysed using flow cytometry. Thirty-five females were identified as carriers by the mosaic expression of Btk and confirmed to be carriers by sequence analysis. Eight females with normal Btk expression, belonging to families with a type I or type II pattern, were confirmed as non-carriers using sequence analysis. One woman, the mother of the only patient (type I pattern) identified in family B-03, showed normal expression of Btk. Sequence analysis revealed only wild-type Btk in her genomic DNA from PBMCs, suggesting that her son is a new mutation. Two other females, belonging to families with type I pattern (families B-18 and B-68), showed normal expression of Btk in both platelets and monocytes. Subsequent sequence analysis identified them as carrier females, suggesting extremely skewed (> 95%) X-chromosome inactivation in favour of the normal X-chromosome. To confirm this interpretation, we evaluated the X-chromosome inactivation status using the human androgen-receptor gene (HUMARA) of two females (patient's mother and sister) in family B-68 (Fig 4). In this family, the mother showed mosaic expression of Btk by flow cytometry, whereas the patient's sister had a normal pattern of Btk expression (Fig 4A). Sequence analysis, however, revealed the existence of a mutated allele in the sisters genomic DNA (Fig 4B). Digestion with the methylation-sensitive endonuclease HpaII showed that only the paternal X-chromosome was activated and that all the maternal X-chromosomes, which carry the mutated Btk gene, were methylated and inactive (Fig 4C). The finding of extremely skewed X-inactivation in the sister explains the normal Btk expression using flow cytometry of both her platelets and monocytes.

image

Figure 4.  Extremely skewed X-chromosome inactivation found in a carrier female of family B-68. (A) Expression of Btk using flow cytometry. Note Btk expression in the sister shows a normal pattern. (B) Sequence analysis of genomic DNA by the dideoxy method using [γ-32P] end-labelled primers. Note that the sister has two alleles, wild and mutated, indicating carrier status. (C) X-chromosome inactivation status of neutrophils as evaluated by DNA methylation of the HUMARA locus. Genomic DNA from each individual was digested with RsaI with or without HpaII. Digested samples were electrophoresed in 7% polyacrylamide gel in non-denatured conditions. The paternal (P) X chromosome of the sister was completely digested with HpaII, indicating complete inactivation of the maternal (M) X chromosome in this individual.

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The other eight females with normal Btk expression belonged to families with a type III pattern and flow cytometric analysis was not informative.

Discussion

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The clinical diagnosis of XLA is based on a history of recurrent bacterial infections starting in infancy, low serum immunoglobulin levels and a decreased number of circulating B cells, with or without a positive family history. The diagnosis is confirmed by demonstrating a mutation within the Btk gene. Mutation analysis, however, requires a specialized laboratory, is time consuming and technically difficult. In some instances, patients with classic XLA were found to have normal sequence analysis of the coding region of Btk, but complete absence or reduced levels of protein (Hashimoto et al, 1996; Gaspar et al, 1998; Holinski-Feder et al, 1998). Alternative methods have been used successfully to establish a molecular diagnosis of XLA in patients with hypogammaglobulinaemia and low B-cell numbers, including in vitro kinase assays (Gaspar et al, 1998), linkage analysis (Kwan et al, 1994) and X-chromosome inactivation studies of obligate XLA carriers (Fearon et al, 1987; Conley & Puck, 1988; Allen et al, 1994). A novel strategy to establish a diagnosis has been introduced by Futatani et al (1998), who demonstrated that XLA patients have normal numbers of peripheral blood monocytes that lack Btk and that carrier females have circulating monocytes that show random X-chromosome inactivation. In contrast, circulating B cells of carriers are known to consistently undergo non-random X-chromosome inactivation (Fearon et al, 1987; Conley & Puck, 1988; Allen et al, 1994). To analyse peripheral blood monocytes for the presence of Btk, the cell membranes were stained with a monocyte-specific (anti-CD14) mAb and the cytoplasm stained with the Btk-specific mAb 48–2H (Futatani et al, 1998).

In this study, we used flow cytometric analysis to evaluate platelets from normal controls and from XLA patients with known mutations for the presence of Btk and to test their female relatives for possible carrier status. The presence of large amounts of Btk in circulating platelets made this analysis possible. As XLA patients have normal platelet counts, a blood sample of less than 5 ml is sufficient to harvest enough platelets for Btk analysis and mononuclear cells for subset analysis, and to isolate mRNA and genomic DNA for sequence analysis. The observation that nearly 100% of normal control platelets express Btk demonstrates that Btk is stable in platelets throughout their lifespan. Although platelets are known to actively synthesize mRNA and protein (Kieffer et al, 1987), very little if any of the platelet-associated Btk is synthesized de novo as eukaryotic mRNA has an average half-life of between 15 min and 17 h (Rajagopalan & Malter, 1997). Thus, most of the platelet-associated Btk must be generated prior to the formation of platelets, which have a lifespan of 8–10 d (Leeksma & Cohen, 1956; Schmidt et al, 1985). The role of Btk for the function of platelets is unknown. Interestingly, XLA patients who lack functional Btk protein owing to mutations of the gene have normal numbers of platelets. Furthermore, there is no disadvantage in the production and survival of Btk-negative platelets as our study has shown that megakaryocytes of carrier females have random X-chromosome inactivation. It has been reported recently that, during platelet activation, Btk is tyrosine phosphorylated. This occurs in platelets that are stimulated with thrombin through engagement of the αIIb3 integrin and activation of phosphoinositide 3-kinase (Laffargue et al, 1999), or with collagen through the collagen receptor (Oda et al, 2000). These observations indicate the involvement of Btk in signalling pathways that are downstream of the adhesive receptors of platelets. Although XLA patients do not suffer from increased haemorrhage, a recent report found diminished responses in platelet aggregation, dense granular secretion and calcium mobilization in XLA platelets, following stimulation through the collagen receptor glycoprotein VI (GPVI) (Quek et al, 1998). The fact that Wiskott–Aldrich syndrome protein (WASP) (which acts downstream of Btk) can be phosphorylated in the absence of Btk further suggests the existence of alternative signalling pathways, e.g. tyrosine kinases such as Tec kinase that bypass Btk in platelets (Oda et al, 2000).

The patients and carrier females included in this study were derived from 45 consecutively seen families with one or more affected males and with known mutations of Btk, and their female relatives. More than half the mutations resulted in single amino acid substitutions. Nonsense mutations, genomic insertions and deletions, and splice site mutations were represented at proportions similar to those observed in the European XLA registry (Vihinen, 1996; Lappalainen et al, 1997; Vihinen et al, 1997). The staining of platelets with mAb 48–2H followed by flow cytometric analysis confirmed the diagnosis of XLA in 37 of the 45 families (78%) with known BtK mutations. Simultaneous staining of circulating monocytes with the same mAb showed identical results, demonstrating that platelets and monocytes are equally useful to screen for Btk mutations. Using the same reagents, a study of Btk expression by monocytes performed in Japan led to the identification of XLA patients in 98% of the families studied (Futatani et al, 1998). This discrepancy is due to a difference in the target population. Of the Japanese group of 41 families, only seven had missense mutations and only one of those had normal expression of Btk (type III). In contrast, in the present study of XLA patients residing in North America, about 50% (22 out of 45) had missense mutations, and affected males in eight of those families showed normal expression of Btk in both platelets and monocytes.

Three patterns of Btk expression were observed in our patient population. A markedly reduced amount or complete absence of Btk (type I) was found predominantly in patients with nonsense mutations, with insertions and deletions resulting in frame shift and premature termination, and in most patients with splice site mutations. Reduced but substantial expression of Btk that allowed differentiation from normal controls (type II) was present in 9 out of 22 families with missense mutations, one out of seven families with nonsense mutations, two out of six families with splice site mutations and in a single family with a mutation affecting the promoter region. Normal or slightly reduced Btk expression (type III) that did not allow differentiation of patients from normal controls or the identification of carrier females using flow cytometry was observed only in families with missense mutations (eight families).

Reduced Btk mRNA or protein expression by PBMCs has been reported in patients with mutations of Btk that resulted in early termination of transcription (Hashimoto et al, 1996; Vorechovsky et al, 1997; Gaspar et al, 1998), possibly owing to the rapid decay of mRNA containing premature stop codons (Peltz et al, 1993; Pulak & Anderson, 1993; Muhlrad & Parker, 1999). The reduced amount of Btk found in 14 of our 22 families with missense mutations is probably as a result of unstable Btk that degrades faster than wild type (Saffran et al, 1994). Gaspar et al (1998) have also observed defective Btk expression in PBMCs from patients with missense mutations by Western blot using polyclonal anti-Btk antibody. Some missense mutations or mutations resulting in in frame deletions or insertions are expected to code for mutated Btk with a reduced affinity for some anti-Btk mAbs but not for others. Such an example is family B-30, which has a mutation within the SH3 domain that causes an in frame deletion of 21 amino acids, resulting in a truncated protein that can be detected by a polyclonal anti-Btk antibody (Zhu et al, 1994), but not by the mAb 48–2H used in this study. Similar findings of discrepancies in results when polyclonal or monoclonal antibodies were used have been observed in X-linked hyper-IgM syndrome (Seyama et al, 1998). As reported by others (Gaspar et al, 1998), there was no recognizable correlation between clinical phenotype and the presence or absence of mutated Btk. The exception was one patient who presented with a mutation in the promoter region of Btk that presumably interferes with the binding of the nuclear protein, PU.1. This patient's platelets and monocytes contained reduced but substantial amounts (type II) of wild-type Btk, which may explain his mild clinical phenotype. De Weers et al (1997) have observed another XLA patient with a mutation within the promoter region and a mild phenotype.

In female carriers of some X-linked conditions such as XLA or X-linked severe combined immunodeficiency (SCID) (Puck et al, 1987; Puck & Willard, 1998), the expected random X inactivation fails to occur in the lymphocyte population targeted by the gene defect. If the gene defect results in a selective disadvantage in the proliferation and survival of the targeted cell lineage, a ‘marked skewing’ of the activation in favour of the X chromosome with the wild-type gene is observed. Such non-random X inactivation has been demonstrated in B lymphocytes of females that are carriers for XLA (Fearon et al, 1987; Conley & Puck, 1988; Allen et al, 1994; Puck & Willard, 1998), but not in their monocytes (Futatani et al, 1998). In this study, we have demonstrated that platelets of female carriers for XLA undergo random X-chromosome inactivation, suggesting that megakaryocytes can develop, mature and generate platelets equally well with or without functional Btk. Consistent with this observation is the finding that XLA carrier females have two populations of platelets, one expressing normal Btk, the other expressing the mutated Btk. The ratio of the two populations varied greatly among female carriers but no tendency of one-directional skewing was observed, further supporting the postulate that absence of Btk does not affect platelet production by megakaryocytes or the survival of circulating platelets.

The expression of two populations of Btk (wild and mutated) during flow cytometry accurately identified carrier females. However, the demonstration of normal Btk expression by a female at risk belonging to an XLA family with a type I or type II pattern does not exclude carrier status. We have identified two females from two unrelated families in which the affected males showed a type I pattern of Btk expression, who both carried the expected mutation (a nonsense mutation and a one base pair deletion respectively) but showed normal Btk expression by their platelets as well as monocytes. This ‘false negative’ result was due to extreme skewed X chromosome inactivation resulting predominantly in the use of the normal X chromosome. Such skewing of X-chromosome inactivation in normal females has been well recognized (Busque et al, 1996; Tonon et al, 1998). A skewing of X-chromosome inactivation of over 90% has been observed in 7% of normal females (Racchi et al, 1998). Thus, it is to be expected that there are female carriers (< 10% in our study) with extremely skewed X-chromosome inactivation in favour of the normal X chromosome, making it impossible to detect the carrier status based on flow cytometric analysis of Btk in peripheral blood platelets or monocytes. For genetic counselling, it is imperative to proceed to mutation analysis in those females at risk of carrier status whose flow cytometric analysis of Btk in platelets shows a normal pattern.

These results demonstrate the usefulness but also the limitation of flow cytometry to analyse Btk expression in platelets as a rapid screening test to identify patients with Btk mutations and carrier females. The simplicity of the test allows the screening of any male with a history of recurrent infections, low serum immunoglobulin levels and decreased numbers of circulating B lymphocytes. If the binding of mAb 48–2H is reduced or absent, the diagnosis of XLA is highly probable, and mutation analysis should be considered as an option to confirm the diagnosis. Because a normal Btk expression does not rule out XLA, as demonstrated in patients with selected missense mutations, sequence analysis is recommended as the final tool. In those families where the affected males express Btk abnormally, carrier females can be identified successfully. The amount of blood required for this analysis is small enough to study infants and, if combined with mutation analysis, can effectively identify affected males and carrier females.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We would like to thank Shigeyuki Arai and his collaborators (Fujisaki Institute, Hayashibara Bio Chemical Laboratories, Okayama) for generating and providing the anti-Btk monoclonal antibody 48–2H.

This study was supported by grant HD 17427 from the National Institute of Health, and grants from the Immune Deficiency Foundation and The Jeffrey Modell Foundation.

References

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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