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Neutropenia in X-Linked Agammaglobulinemia
CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY
Vol. 81, No. 3, December, pp. 271–276, 1996 Article No. 0188
Neutropenia in X-Linked Agammaglobulinemia JASON E. FARRAR,*,† JURG ROHRER,†
AND
MARY ELLEN CONLEY*,†
*Department of Pediatrics, University of Tennessee College of Medicine, Memphis, Tennessee 38103; and †Department of Immunology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105
X-linked agammaglobulinemia (XLA) is usually considered a disorder of B cell development; however, the gene that is defective in XLA encodes a cytoplasmic tyrosine kinase called Btk, that is expressed throughout myeloid as well as B cell differentiation. A review of medical records of patients in whom mutations in Btk had been identified indicated that 13 of 50 patients (26%) had experienced episodes of profound neutropenia. In 12 of the 13 patients, neutropenia was part of the acute illness that precipitated an evaluation for immunodeficiency. These boys were more likely to be less than a year of age at the time of diagnosis and they were less likely to have a family history of immunodeficiency. Neutropenia was associated with staphylococcal or pseudomonas sepsis in 6 of the patients. The duration of neutropenia was variable but was often more than 1 week. Neutropenia was not seen in any patient with XLA receiving intravenous gammaglobulin. Although neutropenia was not associated with any specific mutation in Btk, most of the alterations in this gene in the patients with XLA and neutropenia resulted in the absence of Btk protein or in amino acid substitutions in sites thought to be critical to Btk function. Btk may not be required for neutrophil production under normal circumstances; however, it may play a role in the response to stress. q 1996 Academic Press, Inc.
INTRODUCTION
X-linked agammaglobulinemia (XLA) is usually described as a defect in humoral immunity or B cell development (1–3). Affected males have the onset of recurrent bacterial infections in the first few years of life. Laboratory studies show profound hypogammaglobulinemia and markedly reduced numbers of B cells. However, T cells and phagocytes are generally normal in number and in function (1–3). In heterozygote carriers of XLA, the mothers of affected boys, all B cells are derived from pre-B cells that have the normal X chromosome as the active X (4, 5). B cell precursors from these women that have the X chromosome bearing the XLA mutation as the active X fail to develop, just as pre-B cells from affected boys do not mature into B
cells. In contrast, T cells and neutrophils from carrier women demonstrate a normal, random pattern of X chromosome inactivation. In 1993, two groups showed that XLA is due to mutations in a cytoplasmic tyrosine kinase that is now called Btk (Bruton tyrosine kinase) (6, 7). This 659 amino acid protein consists of several protein–protein interaction domains, including an amino-terminal PH (pleckstrin homology) domain (8), a proline-rich region, and SH2 and SH3 (Src homology 2 and 3) domains, as well as a 269 amino acid carboxy-terminal catalytic domain (6, 7). Although the substrates phosphorylated by Btk have not yet been identified, this enzyme, like other tyrosine kinases, is presumed to function in signal transduction (9). Preliminary evidence suggests that antigen binding to B cells, as mimicked by crosslinking of surface immunoglobulin, is one mechanism to initiate the signal transduction pathway involving Btk because it results in the phosphorylation and increased catalytic activity of this enzyme (10–12). In addition, the protein–protein interaction domains of Btk bind to several proteins known to be involved in signal transduction, including the bg subunits of G proteins (13, 14), protein kinase C (15), cbl (16), and src-related kinases (17). As expected, expression of Btk is limited to hematopoietic cells; however, based on the lack of evidence for phagocytic defects in XLA and the random pattern of X inactivation in neutrophils from carriers, it was a surprise to find that Btk is synthesized in myeloid cells as well as B cells (6, 7). Neutropenia has been reported in patients presumed to have XLA (18–21), usually in critically ill patients at the time of diagnosis. This neutropenia has been attributed to the toxic effects of the infecting organism (21). However, the fact that Btk is expressed in myeloid, as well as B cells, raises the question of whether certain specific mutations in the Btk gene might predispose the patient to neutropenia (22). Three of the 14 patients with XLA receiving care at our institution had a past history of neutropenia. Because we were interested in determining the correlation between the specific mutations in Btk and the clinical consequences of these mutations, we obtained clini-
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cal information on an additional 36 XLA patients for whom we were asked to provide mutation detection. Ten of these 36 patients had a past history of neutropenia. The mutations in Btk in the 13 patients with neutropenia and the clinical and laboratory findings in these patients were compared to those of the entire group. MATERIALS AND METHODS
Patients. Three of the patients included in this study received all of their subspecialty care at St. Jude Children’s Research Hospital or LeBonheur Children’s Hospital in Memphis, Tennessee. The 10 remaining patients were referred by genetic counselors or immunologists from 10 different medical centers for confirmation of the diagnosis of XLA or for carrier detection. The referring physician was asked to complete a clinical survey which included questions about the presenting illness and the incidence of neutropenia. Patients were considered to have neutropenia if they had at least one absolute neutrophil count of less than 500/ mm3 with at least two documented counts of less than 1000/mm3. The duration of neutropenia was defined as the period during which the absolute neutrophil count was less than 1500/mm3. The mutations in Btk in some of these patients were previously reported (23, 24). Mutation detection. Genomic DNA from patients was screened by single-strand conformational polymorphism (SSCP) analysis to localize the defect in Btk to a specific exon. That exon was then amplified in two separate PCR reactions and the PCR products were cloned and sequenced as previously described (23). RESULTS
To determine the incidence of neutropenia in patients with XLA and to delineate the clinical circumstances during which neutropenia occurred, we reviewed the charts of our patients and we asked individuals who had requested mutation detection for XLA since 1993 to provide us with clinical information, including representative blood counts during episodes of neutropenia, and the results of bone marrow examination. Of the 93 members of 70 unrelated families for whom we had identified mutations in Btk, the defective gene in XLA, adequate clinical information was available for 50 patients from 40 families. Thirteen of the 50 patients had a past history of neutropenia. Only 1 of the 13 patients with neutropenia had a family history of immunodeficiency (patient 9.80), whereas 23 of the remaining 37 patients had brothers, cousins, or uncles with hypogammaglobulinemia. Neutropenia was present at the time of the acute illness that precipitated an evaluation for immunodeficiency in 12 of the 13 patients with recorded episodes
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of neutropenia (Table 1). Most of these patients were critically ill, 4 had pseudomonas sepsis, and 2 had staphylococcal sepsis. All 4 of the patients with pseudomonas sepsis had extensive necrotic skin lesions; 2 of these patients had perirectal abscesses that required colonic diversion. Rectal abscesses and colonic diversion occurred in an additional patient (patient 23.70) who was not recognized to have sepsis but who was treated with broad spectrum antibiotics before arriving at the referral center. Viral infections were identified in 4 of the patients, 2 of whom had concurrent bacterial sepsis. The ages of the patients who had neutropenia at the time of diagnosis were highly variable. In the 11 patients with no family history of hypogammaglobulinemia, the mean age at diagnosis was 2.3 years; of note, 6 of these 11 patients were less than 1 year of age at the time of diagnosis. In contrast, of the 20 patients who were the first in their family to be diagnosed as having hypogammaglobulinemia but who did not have a history of neutropenia, only 1 was less than 1 year of age at the time of diagnosis and the mean age at diagnosis was 3.0 years. Three patients were reported as having had more than one episode of neutropenia. In patient 6.00, both episodes of neutropenia occurred when the patient’s serum IgG concentration was less than 200 mg/dl while he was receiving plasma therapy for replacement of gammaglobulin. The second episode of neutropenia in patient 23.00 also happened before he was started on intravenous gammaglobulin. Patient 6.21 was reported as having had neutropenia associated with fever and otitis, 4 years before he was found to have hypogammaglobulinemia. Moderately decreased lymphocyte counts were seen in several patients, particularly those who were critically ill (patients 9.8, 17.00, 20.17, and 22.00). However, the absolute lymphocyte count was greater than 1000/mm3 in all patients. Two patients (patients 22.00 and 23.70) had significant anemia with a hemoglobin of 7.4 g/dl. All other patients had a hemoglobin greater than 10 g/dl. The platelet count was greater than 150,000/mm3 in all of the patients. In only one patient (patient 23.70) was it possible to determine the exact duration of neutropenia. In this patient, serial blood counts were performed which documented both the development and the resolution of neutropenia. The neutrophil count in this patient fell while he was receiving antibiotics but before gammaglobulin therapy was started. The neutrophil count was intermittently low during the first month of gammaglobulin treatment. In the remaining patients, it was not possible to determine when the neutropenia started. Neutropenia resolved promptly after the onset of antibiotic therapy in patients 5.00 and 9.80, despite the fact that they were not recognized to have hypogammaglobulinemia and did not begin receiving gam-
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TABLE 1 Clinical Characteristics of Patients with XLA and Neutropenia Patient No.
Year of birth
Age when neutropenic
Neutropenia at presentation
Duration of neutropenia
WBC
Infection at time of neutropenia
5.00
1990
3 months
Yes
0
3 days
14,100
10 weeks 6 weeks NAa 3 weeks
5,600 8,900 NA 3,400
72
11 days
7,200
Yes
204
2 days
1,700
months months months years years
Yes Yes Yes Yes Yes
110 412 NA 44 98
10 days 1 week 4 weeks NA 5 days
11,000 10,300 3,100 2,200 1,400
1992
25 months
Yes
NA
NA
NA
23.00
1981
23.70
1989
8 months 20 months 18 months
Yes No Yes
220 420 220
2 weeks NA 18 days
11,000 7,000 4,400
Fever; staphlococcal bullous impetigo; adenovirus Fever; otitis; enterovirus Fever, otitis, rash Fever; otitis Pneumonia; impetigo; staphlococcal sepsis Fever; diarrhea; staphlococcal sepsis; rotavirus Pyoderma gangrenosa; pseudomonas sepsis Periorbital cellulitis; impetigo Pseudomonas sepsis Fever Pneumonia Perirectal abscess and pyoderma gangrenosa; pseudomonas sepsis; conlonic diversion; rotavirus Perirectal abscess; pseudomonas sepsis Pneumonia NA Perianal abscess; colonic diversion
6.00
1976
6.21
1988
months years months years
No No No Yes
700 89 0 0
7.10
1990
3 months
Yes
9.80
1994
11 months
14.40 14.71 17.00 20.17 22.00
1993 1992 1978 1981 1978
4 8 10 6 8
22.10
a
24 5 11 5
Lowest ANC/mm3
Not available.
maglobulin therapy until several weeks after admission to the hospital. In patient 6.21, neutropenia persisted for 3 weeks after antibiotics were started and 2 weeks after gammaglobulin therapy was begun. Bone marrow biopsies or aspirates were performed in 4 of the 13 patients. A maturational arrest of the myeloid lineage with normal immature cells was seen in patients 6.00, 9.80, and 23.70. Patient 17.00 had an absolute neutrophil count of 1000/mm3 when he had a bone marrow aspirate that was interpreted as normal. Genomic DNA from the entire group of patients was screened by SSCP analysis to identify mutations in Btk. The mutations found in the patients with neutropenia are shown in Table 2. DNA from patient 6.00 demonstrated a four base pair deletion, resulting in a frameshift mutation with a secondary premature stop codon at codon 120. Single base pair substitutions were seen in the DNA of the remaining 12 patients. Three of these mutations caused premature stop codons and two occurred at invariant sites in the splice donor (patient 20.17) or acceptor (patient 7.10) site. Previous studies have shown that most mutations in Btk causing premature stop codons, frameshifts, or splice defects result in defective accumulation of Btk message in the cytoplasm (23–25). The other 7 patients with XLA and neutropenia had amino acid substitutions, most of which were at sites postulated to be critical to Btk function. Both amino
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acid substitutions in the PH domain were in the region required for binding to the bg subunits of G proteins (13, 14). The lysine at codon 430 is essential for ATP binding (7). The amino acids at codons 509, 581, and 582 are postulated to be important for the structure and stability of the catalytic site (26). In the entire group of 70 unrelated XLA patients in whom mutations have been identified, 10 had small insertions or deletions resulting in frameshift mutations, 2 had three base pair deletions causing the deletion of a single amino acid, 16 had splice defects, 17 had premature stop codons, 1 had a single base pair substitution in the start codon, and 24 had amino acid substitutions. DISCUSSION
This study documents the high incidence of neutropenia as a presenting finding in patients with X-linked agammaglobulinemia. In 24% of patients with XLA, neutropenia was part of the acute illness that precipitated an evaluation for immunodeficiency. This figure is likely to be an underestimate of the true incidence; an episode of neutropenia that occurred in the absence of significant infection or in the distant past may not have been recorded. It is worth noting that 8 of the 13 patients with neutropenia (61%) were diagnosed as having XLA since 1990, whereas 8 of the 37 patients without recorded episodes of neutropenia (22%) were
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TABLE 2 Mutations in Btk in Patients with XLA and Neutropenia Patient No.
Codon
Domain
Change
Effect
5.00 6.00 6.21 7.10 9.80 14.40 14.71 17.00 20.17 22.00 22.10 23.00 23.70
76, 77 113 115 Intron 5 244 430 509 520 Intron 16 581 582 591 622
PH domain PH domain PH domain PH domain SH3 Kinase Kinase Kinase Kinase Kinase Kinase Kinase Kinase
GAAA deletion GTC r GAC TCC r TTC A r G (02) TTG r TAG AAG r AGG ATG r ATA CGA r TGA G r T (/1) TGG r CGG GCT r GTT TAC r TAA GCT r CCT
Frameshift Val r Asp Ser r Phe Splice acceptor defect Leu r Stop Lys r Arg Met r Ile Arg r Stop Spice donor defect Trp r Arg Ala r Val Tyr r Stop Ala r Pro
diagnosed in this time period. In a series of 96 patients with presumed XLA retrospectively analyzed in 1982, Lederman and Winkelstein reported a 10% incidence of neutropenia (20). They also noted that the neutropenia occurred in association with infection. It is doubtful that the incidence of neutropenia in XLA is increasing, although it is possible that more children are surviving an episode of overwhelming infection long enough to permit evaluation for immunodeficiency. The mutations in Btk found in the patients with XLA and neutropenia were highly variable and were representative of the mutations that occurred in the entire group of patients with XLA. Although deletions in Btk, detectable by Southern blot analysis, do occur (7, 27), most mutations found by our group and by others are single base pair substitutions or small insertions or deletions (23–26, 28–34). Past studies have shown that most premature stop codons, frameshift mutations, and splice defects cause a marked reduction or absence of Btk transcript in the cytoplasm (23–25). Thus, these mutations are functionally equivalent in that they result in the absence of Btk protein. Six of the 13 patients with neutropenia and 44 of the entire group of 70 XLA patients had these types of mutations. The remaining patients in both groups had amino acid substitutions or single amino acid deletions, most of which occurred in the catalytic domain of Btk. Attempts to correlate the phenotype of patients with XLA with specific mutations in Btk have not shown strong associations, although amino acid substitutions in the SH2 domain and in regions of the catalytic domain that are not involved in ATP binding, the catalytic site, or substrate binding often appear to be associated with milder disease (28, 29, 35). Severe disease as characterized by persistence of infection may be affected by the presence of chronic lung or ear infections at the time of diagnosis, the compliance with medical therapy, and modifying genetic factors (36). It is note-
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worthy that the patients who had neutropenia at the time of diagnosis have not had unusually severe disease after the onset of intravenous gammaglobulin therapy and, in some cases, prophylactic antibiotics. Neutropenia was not reported in patients who were receiving intravenous gammaglobulin. The etiology of neutropenia in patients with XLA is not clear. Although other defects in antibody production are also associated with neutropenia, the mechanisms are likely to be different. Children with common variable immunodeficiency often have neutropenia, usually in association with thrombocytopenia and/or hemolytic anemia (37). In these patients, autoimmune mechanisms may be the cause of neutropenia. Persistent or recurrent neutropenia is seen in 40–50% of patients with X-linked hyper-IgM syndrome (38), a disorder caused by mutations in the gene for CD40 ligand (39). Although the basis of neutropenia in patients with X-linked hyper-IgM syndrome has not been established, studies in which stimulation of thymic epithelial cells with antibody to CD40 resulted in secretion of GM-CSF (40) provide clues that cytokine dysregulation may play a role. Kozlowski and Evans suggested that the neutropenia in XLA might be due to increased destruction of neutrophils by bacterial toxins (21). However, the overwhelming infections seen in both their patients and our patients, particularly pseudomonas sepsis and staphylococcal sepsis, are typical of those that develop in patients with chronic neutropenia (41, 42), suggesting that the neutropenia in at least some of the XLA patients may have preceded these infections. The coincident occurrence of viral infections in four of the patients brings up the possibility that viral or bacterial infection in these patients induced neutropenia which increased the susceptibility to staphylococcal or pseudomonas infection. Children less than 1 year of age are particularly susceptible to transient neutropenia in association with
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infection. This susceptibility may be exaggerated in patients with XLA. The paucity of mature granulocytic precursors in the bone marrow of patients with XLA and neutropenia suggests inadequate granulocyte production. Although Btk may not be necessary for proliferation or function of myeloid cells under normal circumstances, it may play a role in response to stress. Studies that define the role of Btk in proliferation or differentiation in myeloid cells as well as B cells may resolve this issue. ACKNOWLEDGMENTS We thank the families and referring physicians who participated in this study, Marjorie Fitch-Hilgenburg and Colin Milburn for excellent technical assistance, and Janice Mann for help in preparation of the manuscript. These studies were supported in part by grants from the National Institutes of Health (AI25129), NCI CORE Grant P30 CA21765, American Lebanese Syrian Associated Charities, and funds from the Federal Express Chair of Excellence. REFERENCES 1. Conley, M. E., Parolini, O., Rohrer, J., and Campana, D., Xlinked agammaglobulinemia: New approaches to old questions based on the identification of the defective gene. Immunol. Rev. 138, 5–21, 1994. 2. Smith, C. I. E., Islam, K. B., Vorechovsky, I., Olerup, O., Wallin, E., Hodjattallah, R., Baskin, B., and Hammarstro¨m, L., X-linked agammaglobulinemia and other immunoglobulin deficiencies. Immunol. Rev. 138, 160–183, 1994. 3. Ochs, H. D., and Winkelstein, J., Disorders of the B-cell system. In ‘‘Immunologic Disorders in Infants and Children’’ (E. R. Stiehm, Ed.), pp. 296–338, Saunders, Philadelphia, 1996. 4. Conley, M. E., Brown, P., Pickard, A. R., Buckley, R. H., Miller, D. S., Raskind, W. H., Singer, J. W., and Fialkow, P. J., Expression of the gene defect in X-linked agammaglobulinemia. N. Engl. J. Med. 315, 564–567, 1986. 5. Fearon, E. R., Winkelstein, J. A., Civin, C. I., Pardoll, D. M., and Vogelstein, B., Carrier detection in X-linked agammaglobulinemia by analysis of X-chromosome inactivation. N. Engl. J. Med. 316, 427–431, 1987. 6. Tsukada, S., Saffran, D. C., Rawlings, D. J., Parolini, O., Allen, R. C., Klisak, I., Sparkes, R. S., Kubagawa, H., Mohandas, T., Quan, S., Belmont, J. W., Cooper, M. D., Conley, M. E., and Witte, O. N., Deficient Expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia. Cell 72, 279– 290, 1993. 7. Vetrie, D., Vorechovsky, I., Sideras, P., Holland, J., Davies, A., Flinter, F., Hammarstrom, L., Kinnon, C., Levinsky, R., Bobrow, M., Smith, C. I. E., and Bentley, D. R., The gene involved in Xlinked agammaglobulinemia is a member of the src family of protein–tyrosine kinases. Nature 361, 226–233, 1993. 8. Musacchio, A., Gibson, T., Rice, P., Thompson, J., and Saraste, M., The PH domain: A common piece in the structural patchwork of signalling proteins. Trends Biochem. Sci. 18, 343–348, 1993. 9. Bolen, J. B., Nonreceptor tyrosine protein kinases. Oncogene 8, 2025–2031, 1993. 10. de Weers, M., Brouns, G. S., Hinshelwood, S., Kinnon, C., Schuurman, R. K. B., Hendriks, R. W., and Borst, J., B-cell antigen receptor stimulation activates the human Bruton’s tyrosine kinase, which is deficient in X-linked agammaglobulinemia. J. Biol. Chem. 269, 23,857–23,860, 1994.
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Received April 22, 1996; accepted with revision August 23, 1996
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Vol. 81, No. 3, December, pp. 271–276, 1996 Article No. 0188
Neutropenia in X-Linked Agammaglobulinemia JASON E. FARRAR,*,† JURG ROHRER,†
AND
MARY ELLEN CONLEY*,†
*Department of Pediatrics, University of Tennessee College of Medicine, Memphis, Tennessee 38103; and †Department of Immunology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105
X-linked agammaglobulinemia (XLA) is usually considered a disorder of B cell development; however, the gene that is defective in XLA encodes a cytoplasmic tyrosine kinase called Btk, that is expressed throughout myeloid as well as B cell differentiation. A review of medical records of patients in whom mutations in Btk had been identified indicated that 13 of 50 patients (26%) had experienced episodes of profound neutropenia. In 12 of the 13 patients, neutropenia was part of the acute illness that precipitated an evaluation for immunodeficiency. These boys were more likely to be less than a year of age at the time of diagnosis and they were less likely to have a family history of immunodeficiency. Neutropenia was associated with staphylococcal or pseudomonas sepsis in 6 of the patients. The duration of neutropenia was variable but was often more than 1 week. Neutropenia was not seen in any patient with XLA receiving intravenous gammaglobulin. Although neutropenia was not associated with any specific mutation in Btk, most of the alterations in this gene in the patients with XLA and neutropenia resulted in the absence of Btk protein or in amino acid substitutions in sites thought to be critical to Btk function. Btk may not be required for neutrophil production under normal circumstances; however, it may play a role in the response to stress. q 1996 Academic Press, Inc.
INTRODUCTION
X-linked agammaglobulinemia (XLA) is usually described as a defect in humoral immunity or B cell development (1–3). Affected males have the onset of recurrent bacterial infections in the first few years of life. Laboratory studies show profound hypogammaglobulinemia and markedly reduced numbers of B cells. However, T cells and phagocytes are generally normal in number and in function (1–3). In heterozygote carriers of XLA, the mothers of affected boys, all B cells are derived from pre-B cells that have the normal X chromosome as the active X (4, 5). B cell precursors from these women that have the X chromosome bearing the XLA mutation as the active X fail to develop, just as pre-B cells from affected boys do not mature into B
cells. In contrast, T cells and neutrophils from carrier women demonstrate a normal, random pattern of X chromosome inactivation. In 1993, two groups showed that XLA is due to mutations in a cytoplasmic tyrosine kinase that is now called Btk (Bruton tyrosine kinase) (6, 7). This 659 amino acid protein consists of several protein–protein interaction domains, including an amino-terminal PH (pleckstrin homology) domain (8), a proline-rich region, and SH2 and SH3 (Src homology 2 and 3) domains, as well as a 269 amino acid carboxy-terminal catalytic domain (6, 7). Although the substrates phosphorylated by Btk have not yet been identified, this enzyme, like other tyrosine kinases, is presumed to function in signal transduction (9). Preliminary evidence suggests that antigen binding to B cells, as mimicked by crosslinking of surface immunoglobulin, is one mechanism to initiate the signal transduction pathway involving Btk because it results in the phosphorylation and increased catalytic activity of this enzyme (10–12). In addition, the protein–protein interaction domains of Btk bind to several proteins known to be involved in signal transduction, including the bg subunits of G proteins (13, 14), protein kinase C (15), cbl (16), and src-related kinases (17). As expected, expression of Btk is limited to hematopoietic cells; however, based on the lack of evidence for phagocytic defects in XLA and the random pattern of X inactivation in neutrophils from carriers, it was a surprise to find that Btk is synthesized in myeloid cells as well as B cells (6, 7). Neutropenia has been reported in patients presumed to have XLA (18–21), usually in critically ill patients at the time of diagnosis. This neutropenia has been attributed to the toxic effects of the infecting organism (21). However, the fact that Btk is expressed in myeloid, as well as B cells, raises the question of whether certain specific mutations in the Btk gene might predispose the patient to neutropenia (22). Three of the 14 patients with XLA receiving care at our institution had a past history of neutropenia. Because we were interested in determining the correlation between the specific mutations in Btk and the clinical consequences of these mutations, we obtained clini-
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cal information on an additional 36 XLA patients for whom we were asked to provide mutation detection. Ten of these 36 patients had a past history of neutropenia. The mutations in Btk in the 13 patients with neutropenia and the clinical and laboratory findings in these patients were compared to those of the entire group. MATERIALS AND METHODS
Patients. Three of the patients included in this study received all of their subspecialty care at St. Jude Children’s Research Hospital or LeBonheur Children’s Hospital in Memphis, Tennessee. The 10 remaining patients were referred by genetic counselors or immunologists from 10 different medical centers for confirmation of the diagnosis of XLA or for carrier detection. The referring physician was asked to complete a clinical survey which included questions about the presenting illness and the incidence of neutropenia. Patients were considered to have neutropenia if they had at least one absolute neutrophil count of less than 500/ mm3 with at least two documented counts of less than 1000/mm3. The duration of neutropenia was defined as the period during which the absolute neutrophil count was less than 1500/mm3. The mutations in Btk in some of these patients were previously reported (23, 24). Mutation detection. Genomic DNA from patients was screened by single-strand conformational polymorphism (SSCP) analysis to localize the defect in Btk to a specific exon. That exon was then amplified in two separate PCR reactions and the PCR products were cloned and sequenced as previously described (23). RESULTS
To determine the incidence of neutropenia in patients with XLA and to delineate the clinical circumstances during which neutropenia occurred, we reviewed the charts of our patients and we asked individuals who had requested mutation detection for XLA since 1993 to provide us with clinical information, including representative blood counts during episodes of neutropenia, and the results of bone marrow examination. Of the 93 members of 70 unrelated families for whom we had identified mutations in Btk, the defective gene in XLA, adequate clinical information was available for 50 patients from 40 families. Thirteen of the 50 patients had a past history of neutropenia. Only 1 of the 13 patients with neutropenia had a family history of immunodeficiency (patient 9.80), whereas 23 of the remaining 37 patients had brothers, cousins, or uncles with hypogammaglobulinemia. Neutropenia was present at the time of the acute illness that precipitated an evaluation for immunodeficiency in 12 of the 13 patients with recorded episodes
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of neutropenia (Table 1). Most of these patients were critically ill, 4 had pseudomonas sepsis, and 2 had staphylococcal sepsis. All 4 of the patients with pseudomonas sepsis had extensive necrotic skin lesions; 2 of these patients had perirectal abscesses that required colonic diversion. Rectal abscesses and colonic diversion occurred in an additional patient (patient 23.70) who was not recognized to have sepsis but who was treated with broad spectrum antibiotics before arriving at the referral center. Viral infections were identified in 4 of the patients, 2 of whom had concurrent bacterial sepsis. The ages of the patients who had neutropenia at the time of diagnosis were highly variable. In the 11 patients with no family history of hypogammaglobulinemia, the mean age at diagnosis was 2.3 years; of note, 6 of these 11 patients were less than 1 year of age at the time of diagnosis. In contrast, of the 20 patients who were the first in their family to be diagnosed as having hypogammaglobulinemia but who did not have a history of neutropenia, only 1 was less than 1 year of age at the time of diagnosis and the mean age at diagnosis was 3.0 years. Three patients were reported as having had more than one episode of neutropenia. In patient 6.00, both episodes of neutropenia occurred when the patient’s serum IgG concentration was less than 200 mg/dl while he was receiving plasma therapy for replacement of gammaglobulin. The second episode of neutropenia in patient 23.00 also happened before he was started on intravenous gammaglobulin. Patient 6.21 was reported as having had neutropenia associated with fever and otitis, 4 years before he was found to have hypogammaglobulinemia. Moderately decreased lymphocyte counts were seen in several patients, particularly those who were critically ill (patients 9.8, 17.00, 20.17, and 22.00). However, the absolute lymphocyte count was greater than 1000/mm3 in all patients. Two patients (patients 22.00 and 23.70) had significant anemia with a hemoglobin of 7.4 g/dl. All other patients had a hemoglobin greater than 10 g/dl. The platelet count was greater than 150,000/mm3 in all of the patients. In only one patient (patient 23.70) was it possible to determine the exact duration of neutropenia. In this patient, serial blood counts were performed which documented both the development and the resolution of neutropenia. The neutrophil count in this patient fell while he was receiving antibiotics but before gammaglobulin therapy was started. The neutrophil count was intermittently low during the first month of gammaglobulin treatment. In the remaining patients, it was not possible to determine when the neutropenia started. Neutropenia resolved promptly after the onset of antibiotic therapy in patients 5.00 and 9.80, despite the fact that they were not recognized to have hypogammaglobulinemia and did not begin receiving gam-
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TABLE 1 Clinical Characteristics of Patients with XLA and Neutropenia Patient No.
Year of birth
Age when neutropenic
Neutropenia at presentation
Duration of neutropenia
WBC
Infection at time of neutropenia
5.00
1990
3 months
Yes
0
3 days
14,100
10 weeks 6 weeks NAa 3 weeks
5,600 8,900 NA 3,400
72
11 days
7,200
Yes
204
2 days
1,700
months months months years years
Yes Yes Yes Yes Yes
110 412 NA 44 98
10 days 1 week 4 weeks NA 5 days
11,000 10,300 3,100 2,200 1,400
1992
25 months
Yes
NA
NA
NA
23.00
1981
23.70
1989
8 months 20 months 18 months
Yes No Yes
220 420 220
2 weeks NA 18 days
11,000 7,000 4,400
Fever; staphlococcal bullous impetigo; adenovirus Fever; otitis; enterovirus Fever, otitis, rash Fever; otitis Pneumonia; impetigo; staphlococcal sepsis Fever; diarrhea; staphlococcal sepsis; rotavirus Pyoderma gangrenosa; pseudomonas sepsis Periorbital cellulitis; impetigo Pseudomonas sepsis Fever Pneumonia Perirectal abscess and pyoderma gangrenosa; pseudomonas sepsis; conlonic diversion; rotavirus Perirectal abscess; pseudomonas sepsis Pneumonia NA Perianal abscess; colonic diversion
6.00
1976
6.21
1988
months years months years
No No No Yes
700 89 0 0
7.10
1990
3 months
Yes
9.80
1994
11 months
14.40 14.71 17.00 20.17 22.00
1993 1992 1978 1981 1978
4 8 10 6 8
22.10
a
24 5 11 5
Lowest ANC/mm3
Not available.
maglobulin therapy until several weeks after admission to the hospital. In patient 6.21, neutropenia persisted for 3 weeks after antibiotics were started and 2 weeks after gammaglobulin therapy was begun. Bone marrow biopsies or aspirates were performed in 4 of the 13 patients. A maturational arrest of the myeloid lineage with normal immature cells was seen in patients 6.00, 9.80, and 23.70. Patient 17.00 had an absolute neutrophil count of 1000/mm3 when he had a bone marrow aspirate that was interpreted as normal. Genomic DNA from the entire group of patients was screened by SSCP analysis to identify mutations in Btk. The mutations found in the patients with neutropenia are shown in Table 2. DNA from patient 6.00 demonstrated a four base pair deletion, resulting in a frameshift mutation with a secondary premature stop codon at codon 120. Single base pair substitutions were seen in the DNA of the remaining 12 patients. Three of these mutations caused premature stop codons and two occurred at invariant sites in the splice donor (patient 20.17) or acceptor (patient 7.10) site. Previous studies have shown that most mutations in Btk causing premature stop codons, frameshifts, or splice defects result in defective accumulation of Btk message in the cytoplasm (23–25). The other 7 patients with XLA and neutropenia had amino acid substitutions, most of which were at sites postulated to be critical to Btk function. Both amino
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acid substitutions in the PH domain were in the region required for binding to the bg subunits of G proteins (13, 14). The lysine at codon 430 is essential for ATP binding (7). The amino acids at codons 509, 581, and 582 are postulated to be important for the structure and stability of the catalytic site (26). In the entire group of 70 unrelated XLA patients in whom mutations have been identified, 10 had small insertions or deletions resulting in frameshift mutations, 2 had three base pair deletions causing the deletion of a single amino acid, 16 had splice defects, 17 had premature stop codons, 1 had a single base pair substitution in the start codon, and 24 had amino acid substitutions. DISCUSSION
This study documents the high incidence of neutropenia as a presenting finding in patients with X-linked agammaglobulinemia. In 24% of patients with XLA, neutropenia was part of the acute illness that precipitated an evaluation for immunodeficiency. This figure is likely to be an underestimate of the true incidence; an episode of neutropenia that occurred in the absence of significant infection or in the distant past may not have been recorded. It is worth noting that 8 of the 13 patients with neutropenia (61%) were diagnosed as having XLA since 1990, whereas 8 of the 37 patients without recorded episodes of neutropenia (22%) were
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TABLE 2 Mutations in Btk in Patients with XLA and Neutropenia Patient No.
Codon
Domain
Change
Effect
5.00 6.00 6.21 7.10 9.80 14.40 14.71 17.00 20.17 22.00 22.10 23.00 23.70
76, 77 113 115 Intron 5 244 430 509 520 Intron 16 581 582 591 622
PH domain PH domain PH domain PH domain SH3 Kinase Kinase Kinase Kinase Kinase Kinase Kinase Kinase
GAAA deletion GTC r GAC TCC r TTC A r G (02) TTG r TAG AAG r AGG ATG r ATA CGA r TGA G r T (/1) TGG r CGG GCT r GTT TAC r TAA GCT r CCT
Frameshift Val r Asp Ser r Phe Splice acceptor defect Leu r Stop Lys r Arg Met r Ile Arg r Stop Spice donor defect Trp r Arg Ala r Val Tyr r Stop Ala r Pro
diagnosed in this time period. In a series of 96 patients with presumed XLA retrospectively analyzed in 1982, Lederman and Winkelstein reported a 10% incidence of neutropenia (20). They also noted that the neutropenia occurred in association with infection. It is doubtful that the incidence of neutropenia in XLA is increasing, although it is possible that more children are surviving an episode of overwhelming infection long enough to permit evaluation for immunodeficiency. The mutations in Btk found in the patients with XLA and neutropenia were highly variable and were representative of the mutations that occurred in the entire group of patients with XLA. Although deletions in Btk, detectable by Southern blot analysis, do occur (7, 27), most mutations found by our group and by others are single base pair substitutions or small insertions or deletions (23–26, 28–34). Past studies have shown that most premature stop codons, frameshift mutations, and splice defects cause a marked reduction or absence of Btk transcript in the cytoplasm (23–25). Thus, these mutations are functionally equivalent in that they result in the absence of Btk protein. Six of the 13 patients with neutropenia and 44 of the entire group of 70 XLA patients had these types of mutations. The remaining patients in both groups had amino acid substitutions or single amino acid deletions, most of which occurred in the catalytic domain of Btk. Attempts to correlate the phenotype of patients with XLA with specific mutations in Btk have not shown strong associations, although amino acid substitutions in the SH2 domain and in regions of the catalytic domain that are not involved in ATP binding, the catalytic site, or substrate binding often appear to be associated with milder disease (28, 29, 35). Severe disease as characterized by persistence of infection may be affected by the presence of chronic lung or ear infections at the time of diagnosis, the compliance with medical therapy, and modifying genetic factors (36). It is note-
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worthy that the patients who had neutropenia at the time of diagnosis have not had unusually severe disease after the onset of intravenous gammaglobulin therapy and, in some cases, prophylactic antibiotics. Neutropenia was not reported in patients who were receiving intravenous gammaglobulin. The etiology of neutropenia in patients with XLA is not clear. Although other defects in antibody production are also associated with neutropenia, the mechanisms are likely to be different. Children with common variable immunodeficiency often have neutropenia, usually in association with thrombocytopenia and/or hemolytic anemia (37). In these patients, autoimmune mechanisms may be the cause of neutropenia. Persistent or recurrent neutropenia is seen in 40–50% of patients with X-linked hyper-IgM syndrome (38), a disorder caused by mutations in the gene for CD40 ligand (39). Although the basis of neutropenia in patients with X-linked hyper-IgM syndrome has not been established, studies in which stimulation of thymic epithelial cells with antibody to CD40 resulted in secretion of GM-CSF (40) provide clues that cytokine dysregulation may play a role. Kozlowski and Evans suggested that the neutropenia in XLA might be due to increased destruction of neutrophils by bacterial toxins (21). However, the overwhelming infections seen in both their patients and our patients, particularly pseudomonas sepsis and staphylococcal sepsis, are typical of those that develop in patients with chronic neutropenia (41, 42), suggesting that the neutropenia in at least some of the XLA patients may have preceded these infections. The coincident occurrence of viral infections in four of the patients brings up the possibility that viral or bacterial infection in these patients induced neutropenia which increased the susceptibility to staphylococcal or pseudomonas infection. Children less than 1 year of age are particularly susceptible to transient neutropenia in association with
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infection. This susceptibility may be exaggerated in patients with XLA. The paucity of mature granulocytic precursors in the bone marrow of patients with XLA and neutropenia suggests inadequate granulocyte production. Although Btk may not be necessary for proliferation or function of myeloid cells under normal circumstances, it may play a role in response to stress. Studies that define the role of Btk in proliferation or differentiation in myeloid cells as well as B cells may resolve this issue. ACKNOWLEDGMENTS We thank the families and referring physicians who participated in this study, Marjorie Fitch-Hilgenburg and Colin Milburn for excellent technical assistance, and Janice Mann for help in preparation of the manuscript. These studies were supported in part by grants from the National Institutes of Health (AI25129), NCI CORE Grant P30 CA21765, American Lebanese Syrian Associated Charities, and funds from the Federal Express Chair of Excellence. REFERENCES 1. Conley, M. E., Parolini, O., Rohrer, J., and Campana, D., Xlinked agammaglobulinemia: New approaches to old questions based on the identification of the defective gene. Immunol. Rev. 138, 5–21, 1994. 2. Smith, C. I. E., Islam, K. B., Vorechovsky, I., Olerup, O., Wallin, E., Hodjattallah, R., Baskin, B., and Hammarstro¨m, L., X-linked agammaglobulinemia and other immunoglobulin deficiencies. Immunol. Rev. 138, 160–183, 1994. 3. Ochs, H. D., and Winkelstein, J., Disorders of the B-cell system. In ‘‘Immunologic Disorders in Infants and Children’’ (E. R. Stiehm, Ed.), pp. 296–338, Saunders, Philadelphia, 1996. 4. Conley, M. E., Brown, P., Pickard, A. R., Buckley, R. H., Miller, D. S., Raskind, W. H., Singer, J. W., and Fialkow, P. J., Expression of the gene defect in X-linked agammaglobulinemia. N. Engl. J. Med. 315, 564–567, 1986. 5. Fearon, E. R., Winkelstein, J. A., Civin, C. I., Pardoll, D. M., and Vogelstein, B., Carrier detection in X-linked agammaglobulinemia by analysis of X-chromosome inactivation. N. Engl. J. Med. 316, 427–431, 1987. 6. Tsukada, S., Saffran, D. C., Rawlings, D. J., Parolini, O., Allen, R. C., Klisak, I., Sparkes, R. S., Kubagawa, H., Mohandas, T., Quan, S., Belmont, J. W., Cooper, M. D., Conley, M. E., and Witte, O. N., Deficient Expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia. Cell 72, 279– 290, 1993. 7. Vetrie, D., Vorechovsky, I., Sideras, P., Holland, J., Davies, A., Flinter, F., Hammarstrom, L., Kinnon, C., Levinsky, R., Bobrow, M., Smith, C. I. E., and Bentley, D. R., The gene involved in Xlinked agammaglobulinemia is a member of the src family of protein–tyrosine kinases. Nature 361, 226–233, 1993. 8. Musacchio, A., Gibson, T., Rice, P., Thompson, J., and Saraste, M., The PH domain: A common piece in the structural patchwork of signalling proteins. Trends Biochem. Sci. 18, 343–348, 1993. 9. Bolen, J. B., Nonreceptor tyrosine protein kinases. Oncogene 8, 2025–2031, 1993. 10. de Weers, M., Brouns, G. S., Hinshelwood, S., Kinnon, C., Schuurman, R. K. B., Hendriks, R. W., and Borst, J., B-cell antigen receptor stimulation activates the human Bruton’s tyrosine kinase, which is deficient in X-linked agammaglobulinemia. J. Biol. Chem. 269, 23,857–23,860, 1994.
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Received April 22, 1996; accepted with revision August 23, 1996
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a50b$$$141
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clina
AP: Clin