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X-LINKED AGAMMAGLOBULINEMIA
HUMORAL IMMUNODEFICIENCIES
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X-LINKED AGAMMAGLOBULINEMIA Hubert 8. Gaspar, MD, PhD, and Christine Kinnon, BSc, PhD
B-lymphocyte development is essential for production of functional antibody and humoral immune responses. The mechanisms that regulate this process are complex and tightly regulated. Defects in nonredundant components of this pathway lead to a clinical and immunologic syndrome of congenital agammaglobulinemia with absence of B-cell development. X-linked aga~aglobulinemia(XLA) was the first of these conditions to be described and is the most common cause of congenital agammaglobulinemia. In 1993, abnormalities in the Bruton’s tyrosine kinase (BTK) gene were shown to be the cause of XLA. A considerable amount of research has since been dedicated to understanding the genetic basis and molecular pathogenesis of the disease.
HSTORY XLA was the first human immunodeficiency described for which the underlying defect was clearly identifed. In 1952 Bruton reported the case of an 8-year-old boy who had suffered from recurrent infections, including sepsis caused by pneumococcus of different serotypes. Subsequent analysis of serum by protein electrophoresis revealed a lack of gammaglobulin? Realizing the association between the absence of gammaglobulin and recurrent infections, Bruton started gammaglobulin replacement therapy and demonstrated considerable clinical improvement. Although there was no family history of other affected males in this
From the Moiecular Immunology Unit, Institute of Child Health, University College London, London, United Kingdom IMMUNOLOGY AND ALLERGY CLIMCS OF NORTH AMERICA
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VOLUME 21 NUMBER 1 FEBRUARY 2001
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initial case, subsequent studies revealed a similar clinical phenotype with an X-linked pedigree.=,35 The term agammaglobulinemia, first used by Bruton, continues to be associated with the condition. Although the phenotype of XLA is characterized in its classical form by an almost complete absence of immunoglobulin of all isotypes and the profound reduction of B lymphocytes in the peripheral circulation. Subsequent studies have shown that in some individuals sigruficant amounts of immunoglobulin are detectable in the peripheral blood.5,49 CLINICAL MANIFESTATIONS
The majority of affected boys are prone to recurrent bacterial infection with the peak incidence of onset at 6 months of age following the disappearance of maternal immunoglobulin.4oA study of 96 North American patients found that symptoms appeared in 25% of the patients by the age of 4 months, 50%by 8 months, and 75%by 12 months.4oIn a British study of 44 patients, 40% of patients presented in the first year of life and 21% remained asymptomatic until 3 to 5 years of life.3I In these two studies and another large retrospective the presenting features at diagnosis were variable but the most common clinical problems were pneumonia, otitis media, and gastrointestinal infection (Table 1). Other major infectious problems included arthritis, septicaemia, and meningitis. SPECTRUM OF INFECTIONS
Infections in X U usually are caused by pyogenic bacteria with ~ u e m o p ~ ~ infmzae, lus Staphylococcus aureus, Streptococcus pneumoniae, and Pseudomonas being the most common speciesa although many other types also have been reported.61,70,84 These reports were published before Table 1. INFECTIVE PRESENTATIONS IN PATIENTS WITH X U Numbers (%) of patients with X U in each study Presenting Infection Ear, nose, and throat infection Pneumonia Gastrointestinal infectiOnS Meningitis Septic arthritis Bacterial skin sepsis
Ledefman and WlnkeIsteiP
(n = 96) 72 (75) 54 (56) 34 (35) 10 (10) 8 (8) 27 (28)
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the introduction of the H. influenme B vaccination programs, and although no recent studies have been published, it is likely that the spectrum of infection is now considerably different. Susceptibility to M y c ~ infections ~ ~ ~ remains s ~ ~ a problem in XLA and may cause a generalized systemic infection or be localized to the respiratory tract, joints, or urogenital tract. It is important to note that these infections may be of a more prolonged nature with an insidious onset. Arthritis
Arthritis is a common feature in XLA occurring in approximately 20% of patientsJ6S4OAlthough septic arthritis caused by pyogenic organisms is described, other patients suffer from a nonseptic monoarthritic form that characteristically affects the large joints, resulting in hydrarthrosis and a limited range of movement. There is usually no joint pain or evidence of joint destruction, and there is resolution of symptoms when ~ u n o g l o b therapy u~ is initiated or increased, suggesting an unidentified infectious cause. In addition to pyogenic bacterial and nonseptic forms of arthritis, enterovirus and mycoplasma species have also been identified in synovial fluid from other patients. Chronic infections
Lederman and WinkelsteinM also reported a high incidence of chronic infective complications with chronic pulmonary disease affecting approximately 50% of patients with XLA. The prevalence of pulmonary disease was age related, with 13% of affected patients under 10 years old, 41% between 10 and 20 years old, and 76% greater than 20 years of age. The other major chronic complications were hearing loss (32% of cases) caused by chronic otitis media and meningo-encephalitis and delayed speech acquisition (14% of cases). Enterovirai infections
Resistance to viral infection remains predominantly intact except for a susceptibility to enteroviral infection.45In two major retrospective analyses,3’rrnenteroviral infections were shown to cause a meningoencephalitis sometimes in association with a dermatomyositis-like syndrome. These infections were seen despite gammaglobulin replacement therapy in some individuals. The most. common organism isolated was echovirus, although coxsackie viruses A and B also have been reported to cause disease.45The incidence of enteroviral disease in current populations of XLA patients appears to be less frequent, a finding that may be as a result of improved clinical monitoring of patients and optimal immunoglobulin substitution therapy, This acute sensitivity of patients
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with XLA and other patients with hypogamrnmaglobulinemia to CNS enteroviral infection highlights the need for a thorough investigation of patients with XLA presenting with neurologic symptoms. The pattern of presentation can be variable, with some patients developing sIow progressive neurologic abnormalities including ataxia, paresis, loss of cognitive function, emotional lability, and personality changes. In others the onset is more acute and manifest by fever, headache, and seizures. Examination of the cerebrospinal fluid (CSF) shows a pleocytosis in most cases with a predominance of lymphocytes and a protein concentration typically between 0.5 and 5 g/L.& Culture of enterovirus has historically been difficult, but with the development of polymerase chain reaction (PCR) technologs identification of the causative agent has improved con~iderably.8~ Other Infections
As would be expected from a pure humoral abnormality, cell-mediated immunity remains predominantly intact. Increased frequency and severity of infection caused by nonbacte~alorganisms (other than enterovirus) is not a feature of XLA. Although these infections do occur, they are no more serious than in normal hdividuals and are selflimiting. Exceptions to this include a number of case reports of Pneumocystis carinii pneumonitis in patients with XLA and other hypogammaglobulinemic patients with intact T-cell function.' This suggests that despite the presence of anti-Pnarrnocystis antibodies in pooled immunoglobulin preparations, there may be a susceptibility to Pneumoqsfis infection. XLAANDCANCER
Although certain primary immunodeficiencies can predispose to an increased incidence of tumors (ataxia-telangiectasia, Wiskott-Aldrich syndrome, X-linked lymphoproliferative syndrome), such a relationship is not obvious for XLA. Lederman and Winkelstein documented two patients in their study? one with a B-cell lymphoma of the terminal ileum and the other with a reticulum cell sarcoma of the bowel. One patient with pituitary adenoma was reported by Hermaszewski and Web~ter.~' Analysis of a primary disease registry showed that of 500 patients with neoplasia, 4.2% were reported to have In a study of 52 Dutch patients with XLA, three unrelated young adults were found to have a rapidly progressive colorectal cancer, suggesting that patients with XLA have a 30-fold increased risk of d i e a ~ e This . ~ ~ finding has prompted some authors to propose that complete gastrointestinalstudies should be performed on all XLA patients. Most clinicians, however, reserve such thorough evaluation for those with documented atrophic gastritis where there is a higher risk of gastric cancer.
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MORTALITY
Smith and Witte@ analyzed three major retrospective studies of patients with XLA and found 30 deaths recorded among 170 patients (17.6%). The major cause of death was viral infection and was predominantly caused by disseminated echoviral infection. The other major cause of death was cardiorespiratory failure from chronic pulmonary disease and cor pulmonale. Other causes of death included amyloidosis, septicemia with osteomyelitis, and inflammatory bowel disease. These data are based on analysis of patients who were diagnosed and treated, in many cases, over 30 years ago and most likely do not reflect the current history of the disease. Now that an unambiguous molecular diagnosis can be made, and with improved awareness, antibiotic treatment, monitoring and treatment of the condition, the outlook for individuals with XLA should be much improved. HETEROGENEITY
Identification of BTK as the gene defective in XLA75,77 allows unambiguous assignment of a molecular defect to individuals with abnormalities in antibody production. Although the majority of BTK-deficient patients display the classical immunophenotype of less than 1%peripheral B lymphocytes and virtual absence of all immunoglobulin isotypes, a significant number of atypical or “leaky” phenotypes have been identified, implying that XLA as a disease entity has considerable clinical and immunologic heterogeneity. As many as 20% of patients with mutations in BTK have delayed onset of symptoms or higher concentrations of serum immunoglobulins than expected.” 28, 29, 38, 62 There also appear to be differences in clinical severity in some individuals who have very low concentrations of serum immunoglobulins and almost no B cells but surprisingly few infective 39 The reasons for clinical and immunologic heterogeneity in XLA are unclear and may be dependent on factors other than the BTK defect. IMMUNOPATHOLOGY
The major immunologic abnormality in XLA is the lack of B lymphocytes in the peripheral blood and other organs and the consequent absence of immunoglobulin production. Physical examination of patients with XLA shows only rudimentary adenoidal and tonsillar tissue and peripheral lymphoid hypoplasia. Histologic analysis of secondary lymphoid tissue demonstrates lack of germinal center and follicle formation.= In the lamina propria of the gut, plasma cells typically are absent. Examination of XLA bone marrow reveals a block in B-cell maturation that is variable with regard to the stage of B-cell maturation and severity of the developmentalarrest. In a study of eight patients, analysis
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of bone marrow demonstrated a maturation arrest in the transition between early pre-B cells and pre-B cells? The severity of this block was variable with some patients, showing a normal number of pre-B cells in the bone marrow and suggesting that a later arrest in development was present in these individuals. In subsequent studies, it also has been shown that a differentiation block can occur earlier at the pro- to pre-Bcell transition,& although this block was not absolute (Fig. 1).Detailed analysis of peripheral blood from patients with XLA presenting classical phenotypes reveals that between 0.01% and 0.1% of CD19+ cells can still be detected.& Functional analysis of this B-cell population shows that these cells proliferate and produce i ~ u n o g l o b u l i nE (IgE) in response to CD40 and interleukin-4 (IL-4) stimulation, suggesting that cells that escape the developmental block are still capable of further maturati0n.4~ MOLECULAR BASIS OF XLA
The genetic defect in XLA was identified in 1993 by two groups, one using a positional cloning approachn and the other searching for novel protein kinases expressed in B lymphocyte^.^^ The gene was designated BTK and is located on the long arm of the X chromosome at Xq21.3. The human gene encompasses 37.5kb, is organized into 19 exens,=, 52, 66 including a 5’ untranslated region (exon l), and encodes a 659 amino acid protein. BTK expression is primarily restricted to hematopoietic cells and is found throughout B-cell differentiation from CD34’ pro-B cells to mature B cells but is down-regulated in antibody secreting plasma cells. Expression also is present in the monocytes, 67 There is, myeloid cells, erythrocyte precursors and megakaryocytes.22, however, no evidence of BTK expression in T-cell lineage or in natural killer (NK) cells. The Btk protein belongs to a family of cytoplasmic tyrosine kinases that are related to, but distinct from, the Src family kinases (Fig. 2). The protein is characterized by its modular structure that includes an aminoterminal pleckstrin homology (PH) domain followed by a tec-homology (TH) domain (which includes a proline rich region), an Src homology SH3 domain, an SH2 domain, and a carboxy-terminal kinase SH1 kinase domain. Catalytic activity resides in the kinase domain while the other domains are necessary for protein-protein interactions. Other members of this family include Tec,& Itk,” 87 and Bmx,” all of which are also expressed in hematopoietic tissues. Several key features distinguish the Btk/Tec family of proteins from the Src family kinases. Btk lacks the a ~ o - t e r m i n amyristylation l signal essential for post-translational modification and the membrane localization of S r ~ . At 5 ~ the a m i n ~ t e r m end ~ l of Btk is the PH domain that consists of seven antiparallel p sheets that form a putative ligand binding pocket on one face of the molecule. This pocket has been shown to bind specifically to phosphatidylinositols and phosphatidyl inositol-3,4,5-trisphosphate (PIP3)?4 Thus, in resting cells, Btk is localized in the cyto-
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BtklTec family
src family
SywzAP-70 family
Figure 2. Btk is related to but distinct from the Src family of tyrosine kinases. It shares kinase, SH2, and SH3 domains but has a unique amino-terminal PH and TH domain.
plasm, and membrane recruitment of Btk is mediated by a Btk-PH domain and PIP, interaction. The C-terminal portion of the PH domain contains a highly conserved tryptophan residue, is folded into an ahelix, and can modulate binding to the p.v subunit of heterotrimeric G pr~teins.’~ Btk also lacks the carboxy-terminal tyrosine residue kinases (Y-527in Src) that negatively regulate the catalytic activity of Src kinases? Regulation of Btk catalytic activity is more complex, as demonstrated by the structurally homologous T-cell kinase Itk which shows an intramolecular association of the SH3 domain with the proline rich region of the TH domain thus restricting the access of the SH3 domain to potential Phosphorylation of a tyrosine residue within the SH3 domain may disrupt SH3-TH interaction thereby liberating SH3 and TH domains to form associations with alternative ligands. A summary of the interactions and functions of Btk is illustrated in Table 2. BTK FUNCTION AND ITS ROLE IN B-CELL DEVELOPMENT
Tyrosine kinases have been studied in many hematopoietic cell lineages and have been shown to act as signal transduction molecules mediating cell surface receptor activation events to downstream pathways. Cross-linking of a number of cell surface receptors, including interleukin-5 (IL-5), IL-6, CD38, FcRe, and most importantly surface IgM on B cells, results in the recruitment of cytosolic Btk to the plasma membrane and activation of Btk by tyrosine phosphorylation.41The process of Btk activation is initiated by phosphorylation of tyrosine 551 (Tyr551) in the kinase domain of Btk by an Src family kinase, most likely Lyn, followed by Btk autophosphorylation at tyrosine 223 (Tyr223) in
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the SH3 domain.53*56*82 Btk is then recruited to the cell membrane, a process depends on an intact PH domain and its association with PIP3.16,= At the cell membrane, activated Btk interacts with phospholipase-y2 (PLC-y2) leading to enhanced tyrosine phosphorylation of PLC-y2 and resulting in accumulation of inositol 1,4,5-triphosphate (IP3) and release of calcium from internal stores. This pathway has been well defined in in vitro models13,63 and is supported by evidence from Btk-deficient cell lines that show reduced phosphorylation of PLC-y2 and decreased 71 The detailed molecular mechanisms through calcium which Btk interacts with PLC-y2 are being defined. An adapter molecule, the B-cell-linker protein (BLNK), has been identified that mediates Btk interactions with PLC-y2.15 Following B cell receptor (BCR) crosslinking, activated Syk phosphorylates BLNK on a number of tyrosine residues.15 BLNK is then able to bind through phosphorylated tyrosine motifs to the PLC-y S H 2 domains15 and also to the S H 2 domain of Btk.27 Thus, BLNK may act to bring Btk in close proximity with PLC-y2 and allow Btk phosphorylation of PLC-y2 (Fig. 3). This is not an absolute mechanism because Btk is able to bind to unphosphorylated BLNK and PLC-y2 phosphorylation can occur in the absence of BLNK.2' Optimal function is only seen if phosphorylated BLNK is present, however. The crucial role of BLNK in B-cell development is further illustrated by the phenotypes of BLNK human and murine mutants that show distinct similarity to the respective Btk mutant phenotypes.48, Other biochemical consequences of Btk activation include coimmunoprecipitation with and phosphorylation of a protein termed Btk-associated protein-135 (BAP-135),= which has been independently identified as a DNA-binding protein TFII and thus links Btk with regulation of transcriptional e~ents.2~.Despite the large volume of biochemical and signaling data available, a relationship with cellular events and specifically the mechanism of B-cell development arrest has yet to be established. Clearly, Btk participates in a signal cascade responsible for the mobilization of calcium, but this can initiate several types of transcriptional events and growth responses such as proliferation and apoptosis. The close homology between Btk and the Src family kinases initially suggested that dysregulated Btk may have oncogenic potential. This finding was supported by random retroviral-mediated mutagenesis studies which identified a specific PH mutation that can lead to mutant Btk with enhanced transforming ability." Expression of the E41K mutant (termed Btk*) in NIH3T3 cells resulted in growth of cells in soft agar and was accompanied by an increase in membrane association and tyrosine phosphorylation at Y551 and Y223, suggesting increased activation. A mutation within the SH3 domain potentiated the transforming potential of Btk,* supporting the idea that the SH3 domain has a negative regulatory function. MUTATION ANALYSIS IN XLA
Identification of the BTK gene has led to research in mutational analysis by a number of groups worldwide. Over 500 unique mutations
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in BTK have been identified, and an international database of mutations and clinical i n f o ~ a t i o nhas been established (BTKbase web page: http:/ /www.uta.fi/laitokset/imt/bi~info/BTKbase).~~~ 79, Various different types of genetic a b n o ~ ~ t ihave e s been found in the BTK gene. One third of mutations are missense mutations, and these have been found p r e d o m i n ~ ~ inythe kinase domain of the gene, although they have been found in all domains except the SH3 domain.s0, Premature stop codons, deletions, and insertions also have been described throughout the gene. Although the large majority of mutations are found in the coding region, 12% of mutations have been found to affect splice site recognition sequence^.^ Only one mutation has been found in the promoter region, and this mutation is thought to affect transcriptional regulation of the About 5% to 10% of mutations result in gross alterations of the BTK gene.59In addition to large deletions, an inversion, a duplication, and a retroviral insertion have been identified. Despite the large quantity of mutation and phenotypic data available, it has not been possible to make definitive genotypephenotype correlations. The strongest argument for this comes from reports of considerable variation in clinical severity even within the same pedigree13,39suggesting that factors other than the genetic mutation may influence the clinical course. It has been suggested that, as in other i~nmunodeficiencies,’~ the presence of cytokine gene polymorphisms or defects in innate immunity caused by mutations in m ~ a n - b i n d i n g lectin may be important. There is, however, no evidence to refute or confirm these ideas. It also is possible that environmental factors may play an important role. The consequence of the gene mutation on expression of the Btk protein and its relation to clinical severity also has been explored. It has been found that in the majority of individuals, despite the severity of the clinical phenotype and regardless of the nature of the mutation, there is a complete lack of Btk expression.17,22, 29 It would appear that even c ~ b o x y - t e ~ i namino al acid substitutions render the messenger RNA (mRNA) or Btk protein unstable. A N I ~ A LMODELS
The consequence of mutations in BTK also have been studied in murine models of the disease. The X-linked immunodeficiency (xid) mouse has a single amino acid substitution (R28C) in the PH domain of Btk.55,73 The immunophenotype of the xid mouse shows normal serum concentrations of IgG1, IgG2a and IgGZb but markedly reduced 1 6 3 and IgM concentrations. Mice also are unable to make antibody responses to T-cell-independent antigens but respond well to stimulation with T-cell-dependent antigens.85The block in B-cell maturation is much less severe in xid mice compared with patients with XLA. Whereas typical patients with XLA have less than 1% of the normal number of peripheral B cells, B-cell numbers in xid mice are only reduced to 30% to 50% of the normal amount. In both species, defects in BTK are associated with an immature B-cell phenotype with the majority of B
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cells dispaying an increased density of surface IgM.” 85 The differences between the murine and human phenotypes cannot be explained by the nature of the xid genetic defect because human R28C mutations with severe phenotypes have been de~cribed.~ Furthermore, Btk-null mice created by homologous recombination also have a B-cell phenotype that 36,37 is identical to the xid DIAGNOSIS
The diagnostic criteria for XLA agreed on by members of the PanAmerican Group for Immunodeficiency (PAGTT))and the European Society for ImmundeficiencyS (ESID) are listed as follows: Definitive Male patient with CD19+ B cells less than 2% and at least one of the following: Mutation in BTK Absent BTK mRNA or protein in peripheral blood mononuclear cells or neutrophils Maternal family history of agammaglobulinemiaand abnormal Bcell numbers Probable Male patient with CD19+ B cells less than 2% in whom all of the following are positive: Onset of recurrent bacterial infections in the first 5 years of life Serum IgG, IgM, and IgA more than 2 standard deviations below normal for age Absent isohemagglutinins or poor vaccine responses Other causes of hypogammaglobulinemia have been excluded Possible Male patient with CD19+ B cells less than 2% in whom other causes of hypogammaglobulinemiahave been excluded and at least one of the following is positive: Onset of recurrent bacterial infections in the first 5 years of life %nun IgG, IgM and IgA more than 2SD below normal for age Absent isohemagglutinins or poor vaccine responses Atypical presentations 15% to 20% of patients have higher concentrations of serum immunoglobulins than expected or are not recognized until after 5 years of age Differential diagnosis p heavy chain deficiency A5 surrogate light chain deficiency Iga deficiency BLNK deficiency
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A pattern of X-linked inheritance, severely decreased numbers of peripheral B lymphocytes, and decreased immunoglobulin production makes a clinical diagnosis of XLA very likely. Because approximately one third to one half of XLA cases are sporadic and with the identification of genetic defects are giving rise to autosomal recessive forms of the condition, alternative mechanisms of diagnosis are necessary. There is considerable heterogeneity to the XLA phenotype, as previously discussed, making a diagnosis based on clinical and immunologic criteria difficult. Identification of BTK as the defective gene in XLA allows mutation analysis to be performed, can offer an unambiguous molecular diagnosis, and offers possibilities for carrier assignment and prenatal diagnosis. Mutation analysis for XLA normally involves screening the 19 exons of the BTK gene in genomic DNA by one of many techniques of which single-stranded confirmational polymorphism (SSCP) analysis is the most commonly used before direct sequencing of the affected exon. SSCP analysis can be a time consuming and expensive procedure, and a definitive diagnosis may take weeks to achieve. Furthermore, some mutations may not be detected by SSCP because the sensitivity of the technique is only 85% to 90%. Other screening techniques do exist, but the sensitivities are not necessarily greater than that of SSCP. Examination of Btk protein expression in peripheral blood monocytes by either Western blot or flow cytometric analysis offers a more rapid and equally sensitive diagnostic assay. In flow cytometry studies, 40 of 41 patients were shown to lack Btk expression,17and the diagnosis was made in 10 individuals for whom no previous genetic data was available. In the authors' own experience, 29 of 31 patients had abnormal expression by Western blot analysis with the majority of patients completely lacking expression and two cases showing the presence of abnormally si~ed-protein'~ (and unpublished data). Thus, over 90% of patients with XLA have abnormalities in Btk expression. In the authors' immunodeficiency diagnostic service, the routine strategy is to screen initially by immunoblotting and then refer abnormal findings for mutation analysis. Thus, a rapid diagnosis can be established, and SSCP analysis can be undertaken with greater confidence. If Btk expression is normal, clinical and immunologic findings dictate when referral for genetic testing is appropriate. Immunoblot expression assays are not routinely of use in determining carrier status because of the absence of protein in most patients; however, in one patient in whom a truncated protein was found caused by a frameshift mutation, immunoblot analysis of cells from the mother showed the presence of an abnormal and normal band, confirming her carrier status (unpublished data). The flow cytometric analysis assay does offer the possibility of carrier testing as monocytes from obligate carriers of patients with no Btk expression show a dual population of Btk-expressing and nonexpressing cells.17 The differential diagnosis of XLA, especially in those patients lacking an X-linked pedigree, has increased significantly. Congential agammaglobulinemia with absent B cells has been shown to arise as a result A 5 surrogate of defects in at least four other genes (i.e., p heavy ~hain,8~
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light chain:7 Iga& BLNK&)C, all of which are essential to ~ c t i o n i n g through the B- or pre-B-cell receptor complex. Males with these defects are clinically indistinguishable from those with XLA, although in p and IgoL chain deficiency., examination of B-cell precursors in the bone marrow shows there is a more stringent block in B-cell development at the pro- to pre-&ell transition.&It must be remembered, however, that these autosomal defects are very rare. In a recent analysis of a large chort of males with presumed XLA, 90% to 95% had mutations in BTK? A rationale for diagnosis would be initially to undertake protein and genetic analysis for BTK defects. If no BTK defect was identified, bone marrow sample analysis by RT-PCR and flow cytometry for examining the expression of these candidate genes may prove useful in determining the specific defect. CLINICAL MANAGEMENT
The mainstay of treatment in XLA is immunglobulin replacement therapy. In most institutions, replacement is given on a regular basis through intravenous immunoglobulin (IVIG),but difficulty with venous access in young children and ease of administration has led to some centers adopting subcutaneous administration (SCIG) as an alternative approach. The use of IVIG replacement is now routine in most centers. Treatment aims to achieve serum IgG levels in the normal age-related range. For most patients, treatment involves a dose of approximately 0.4 to 0.6 g/kg every 3 to 4 weeks. Some centers practice a loading dose of 1 g/kg at the initiation of therapy. Dose and frequency should be tailored to the trough levels of immunoglobulin and clinical symptoms. In certain adult centers the practice is to keep levels above 8 g/L for individuals who continue to have recurrent infective problems. This practice may be especially important for the prevention of chronic lung disease. Once established on a regular regime, patients can be trained to self-cannulate and administer M G at home, leading to greater patient convenience. Adverse reactions can be associated with the infusion of IVIG, most commonly including headaches, nausea, vomiting, flushing, and m~algia.6~ More severe symptoms, such as chest pain and anaphylactoid reactions, have been described but are rare. Thus, IVIG should only be given under the supervision of experienced practitioners, and patients on home therapy should be taught to recognize and manage such events. Rapid subcutaneous infusion of immunoglobulin is an alternative o n has many advantages. First used in adult method of a ~ i s ~ a t i that patients with common variable immunodeficiency it is useful in the pediatric population in whom regular venous access can be a difficult and traumatic process. SCIG allows ease of access and is more readily transferable to home a d ~ t r a t i o n Early . studies also suggest that there is no difference in the maintenance of optimal immunoglobulin levelsz0;however, only a limited volume of immunoglobulin can be administered in one infusion, increasing the frequency of infusions,
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especially in older children. Local adverse reactions also have been observed in a few children. Only a concentrated intramuscular preparation, which has not undergone the virus inactivation steps of IV preparations, has been available for use to date, A trial of a specific virus inactivated subcutaneouspreparation currently is underway and demonstration of efficacy with this product may lead to increasing the use of this mode of immunoglobulin administration. Despite the excellent clinical response of most patients to immunoglobulin replacement therapy, it is still important to monitor patients on a regular basis. It is recommended that trough immunoglobulin levels are monitored regularly to ensure that optimal levels are maintained. Liver function tests also should be checked regularly so that any hepatic illness caused by viral transmission can be detected at an early stage. In the authors' practice, all children undergo a chest radiograph or chest CT scan on a yearly basis, as nonsymptomatic infections can occur and can lead to chronic changes. Early detection of such abnormalities can prevent further progression by optimizing replacement therapy or by use of prophylactic antibiotics. Enteroviral disease and especially chronic mening~nc~halitis remain the greatest dangers to patients with XLA and other agammaglobulinemic patients. Effective antiviral agents ha3e not been previously available, but recent reports suggest that pleconaril, an orally active broad-spectrum antipicomaviral agent with excellent penetration of the central nervous system and nasal epithelium, may have significant antienteroviral activity. Studies show that pleconaril has sigruficant activity against the common forms of enteroviral strains in in vitro and in vivo murine models." In an experimentally induced human model of coxsackie A21 respiratory infection, prophylactic use of pleconaril resulted in significant reductions in viral shedding in nasal secretions, nasal mucus production, and total respiratory illness score^.^ One case report has documented its efficacy in treating enteroviral meningoencephalitis by reduction of CSF inflammatory markers and resolution of clinical signs and symptoms.* Its use currently is being piloted in immunodeficient patients, but published results are not yet available. GENE THERAPY
Gene therapy for XLA appears to be an attractive concept as only the B-cell lineage needs to be targeted. Because female carriers show nonrandom X-inactivation in B cells? l1 it would appear that there would be a survival advantage for corrected cells. Transgenic mice experiments have demonstrated the principle that BTK gene expression can overcome the B-cell developmental defects seen in BTK null mice.lO, In one strategy, a yeast artificial chromosome (YAC) transgenic mouse strain was generated in which high level expression of human BTK was provided by endogenous regulatory cis-acting elements present on a 340-kb transgene. When the YAC-btk transgenic mice were mated with the
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BTK- strain,3O BTK- mice with a YAC-btk background were generated. Analysis of the immunophenotype in this population demonstrated restoration of normal numbers of mature B cells in the bone marrow and periphery and reconstitution of the CD5+ B-cell population in the peritoneum. The expression of the YAC-btk transgene restored serum Ig levels and responses to T-cell independent antigens in BTK- mice. In an alternative strategy, a transgenic mouse strain was generated in which expression of the human BTK gene (complimentary DNA [cDNA]) was placed under the control of the murine class 11 major histocompatibility complex Ea gene locus control region, which provides gene expression from the pre-B cell stage onwards.l0 Again, when these mice were mated with the btk- strain, the presence of the transgene on the BTKbackground resulted in the rescue of the btk- strain immune deficits. The success of this latter experiment suggests that the BTK gene does not necessarily need to be introduced into hematopoietic stem cells or pro-B cell populations to restore B-cell development, and tight regulation of BTK expression is not necessary for successful reconstitution. In more recent competitive reconstitution experiments, xid mice defects were corrected when sublethally irradiated mice received a mixture of 25,000 normal cells and 475,000 xid cells.%The identity of cells responsible for reconstitution in this study was shown to be early hematopoietic presursors (CD19- B220 - ) that had not yet committed to the B-cell lineage. Reconstitution was achieved with as little as 10% of normal cells, suggesting that relatively inefficient gene transfer may be successful because of the strong survival advantage for normal cells. SUMMARY
X-linked agammaglobulinemia remains the prototypic humoral immunodeficiency with absence of immunoglobulin production caused by an abnormality in B-cell development. Identification of BTK as the defective gene has led to a greater understanding of the molecular mechanisms involved in signaling downstream of B-cell surface receptors, although the pathogenesis of the arrest in B-cell development remains to be determined. Identification of BTK also has led to improved methods of diagnosis and carrier detection and has demonstrated the clinical heterogeneity of BTK abnormalities. This raises further questions regarding the factors that ultimately determine the severity of the clinical phenotype. Clinical management of XLA has been dependent on gammaglobulin substitution, and a variety of preparations and modes of administration are now available. The outlook for patients with XLA is good as long as patients are carefully monitored and maintained on adequate immunoglobulin substitution. With the development of effective antienteroviral therapy, the major cause of mortality in XLA may soon be treatable. Observations from carrier females and murine studies all suggest that XLA may be a good candidate for gene therapy. There is a strong selective advantage for corrected cells, and tight regulation
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of gene transfer is not a necessity. Thus, new possibilities for treatment may soon become available.
References 1. Bonagura VR, Cunningham-RundlesS, Edwards BL, et al: Common variable hypogammaglobulinemia, recurrent Pneumocystis curinii pneumonia on intravenous gammaglobulin therapy, and natural killer deficiency. Clin Immunol Immunopathol51:216,1989 2. Bruton OC: Agammaglobulinaemia. Pediatrics 9:722,1952 3. Bykowsky MJ, Haire RN, Ohta Y et al: Discordant phenotype in siblings with X-linked agammaglobulinemia. Am J Hum Genet 58477,1996 4. Campana D, Farrant J, Inamdar N, et ak Phenotypic features and proliferative activity of B cell progenitors in X-linked agammaglobulinemia. J Immunol 1451675,1990 5. Conley ME: B cells in patients with X-linked agammaglobulinemia. J Immunol 134:3070,1985 6. Conley ME, Brown P, Pickard AR, et al: Expression of the gene defect in X - l i e d agammaglobulinemia. N Engl J Med 315564,1986 7. Conley ME, Mathias D, Treadaway, J et al: Mutations in BTK in patients with presumed X-linked agammaglobulinemia. Am J Hum Genet 621034,1998 8. Conley ME, Notarangelo LD, Etzioni A: Diagnostic criteria for primary immunodeficiencies: Representing PAGID (Pan-American Group for Immunodeficiency) and ESID (European Society for Immunodeficiencies). Clin Immunol93190,1999 9. Cooper JA, Howell B: The when and how of Src regulation. Cell 731051,1993 10. Drabek D, Raguz S, De-Wit T l' et al: Correction of the X-linked immunodeficiency phenotype by transgenic expression of human Bruton tyrosine kinase under the control of the class II major histocompatibility complex Ea locus control region. Proc Natl Acad Sci U S A 94610,1997 11. Fearon ER, Winkelstein JA, Civin CI, et a1 Camer deteckon in X-linked agammaglobulinemia by analysis of X-chromosome inactivation. N Engl J Med 316427,1987 12. Filipovich, AH, Shapiro Rs: Tumours in patients with common variable immunodeficiency. J Immunol Immunopharmacol11:43,1991 13. Fluckiger AC, Li Z, Kato RM, et al: Btk/Tec kinases regulate sustained increases in intracellular Ca2' foliowing B-cell receptor activation. EMBO J 17:1973, 1998 14. Foster CB, Lehmbecher T, Mol F, et al: Host defense molecule polymorphisms influence the risk for immune-mediated complications in chronic granulomatous disease. J Clin Invest 1022146,1998 15. Fu C, Turck CW, Kurosaki T, et a1 BLNK a central linker protein in B-cell activation. Immunity 993,1998 16. Fukuda M, Kojima T, Kabayama H, et al: Mutation of the plecksfxin homology domain of Bruton's tyrosine b a s e in immunodeficiency impaired inositol 1,3,4,S-tetrakisphosphate binding capacity. J Biol Chem 271:30303, 1996 17. Futatani T, Miyawaki T, Tsukada S, et a 1 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 camer detection. Blood 91:595,1998 18. Gardulf A, Andersen V, Bjorkander J, et al: Subcutaneousimmunoglobulinreplacement in patients with primary antibody deficiencies: Safety and costs. Lancet 345,365,1995 19. Gaspar HB, Lester T, Levinsky RJ, et al: Bruton's tyrosine kinase expression and activity in X-linked agammaglobulinaemia (XLA): The use of protein analysis as a diagnostic indicator of XLA. Clin Exp Immunol 111:334, 1998 20. Gaspar J, Gemtsen B, Jones A. Immunoglobulin replacement treatment by rapid subcutaneous infusion. Arch Dis Child 7948,1998 21. Genevier HC, Callard RE: Impaired Ca2+mobilization by X-linked agammaglobulinaemia (XLA) B cells in response to ligation of the 8-cell receptor (BCR). Clin Exp Immunol110386,1997 22. Genevier HC, Hinshelwood S, Gaspar HB, et ak Expression of Bruton's tyrosine kinase protein within the B-cell lineage. Eur J Immunol243300,1994
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23. Good RA: Clinical investigations in patients with agammaglobulinemia. J Lab Clin Med 44:803,1954 24. Grueneberg DA, Henry RW, Brauer A, et al: A m u l ~ ~ o nDNA-binding a l protein that promotes the formation of serum response f a ~ o r / h o m e ~ o m acomplexes: in Identity to TFII-I. Genes Dev 11:2482, 1997 25. Hagemann TL, Chen Y, Rosen FS, et a1 Genomic organization of the BTK gene and exon scanning for mutations in patients with X-linked agammaglobulinemia. Hum Mol Genet 3:1743, 1994 26. Hansel TT, Haeney MR, Thompson RA: Primary hypogammaglobulinaemiaand arthritis. Br Med J (Clin Res Ed) 295174,1987 27. Hashimoto S, Iwamatsu A, Ishiai M, et a1 Identification of the SH2 domain binding protein of Bruton's tyrosine kinase as BLNK-Functional significance of Btk-SH2 domain in B-cell antigen receptor-coupled calcium signaling. Blood 942357,1999 28. Hashimoto S, Miyawaki T, Futatani T, et ak Atypical X-linked agammaglobulinemia diagnosed in three adults. Intern Med 38:722, 1999 29. Hashimoto S, Tsukada S, Matsushita M, et al: Identification of Bruton's tyrosine kinase (BTN gene mutations and characterization of the derived proteins in 35 X-linked agammaglobulinemia families: A nationwide study of Btk deficiency in Japan. Blood 88561,1996 30.Hendriks RW, de-Bruijn MF, Maas A, et a1 Inactivation of BTK by insertion of lacZ reveals defects in B-cell development only past the pre-B cell stage. EMBO J 154862,1996 31. Hermaszewski RA, Webster AD Primary hypogammaglobulinaemia: A survey of clinical manifestationsand complications. Q J Med 86:31,1993 32. Heyeck SD, Berg LJ: Developmental regulation of a murine T-cell-specific tyrosine kinase gene, Tsk. Proc Natl Acad Sci U S A 90669,1993 33. H o h k i FE, Weiss M, Brandau 0, et al: Mutation screening of the BTK gene in 56 families with X-linked agammaglobulinemia (XLA): 47 unique mutations without correlation to clinical course. PediaMcs 101:276,1998 34. Hyvonen M, Saraste M Structure of the PH domain and Btk motif from Bruton's tyrosine kinase: Molecular explanations for X-linked agammaglobulinaemia.EMBO J 163396,1997 35. Janeway CA, Apt L, Gitlin D: Agammaglob~emia.Trans Assoc Am Physicians 66200,1953 36. Kerner JD, Appleby MW, Mohr RN, et a1 Impaired expansion of mouse B-cell progenitors lacking Btk. Immunity 3301, 1995 37. Khan WN, Alt FW,Gerstein Rh4, et al: Defective B-cell development and function in Btk-deficient mice. Immunity 3283,1995 38. Kornfeld SJ, Good RA, Litman GW. Atypical X-linked agammaglobulinemia fletter; comment]. N Engl J Med 331:949,1994 39. Kornfeld SJ, H a h RN, Strong SJ,et al: Extreme variation in X-linked agammaglobulinemia phenotype in a three-generationfamily. J Allergy Clin Immunol100702,1997 40. Lederman HM, Winkelstein J A X-linked agammaglobulinemia: An analysis of 96 patients. Medicine (Baltimore) 64:145,1985 41. Li T, Rawlings DJ, Park H, et al: Constitutive membrane association potentiates activation of Bruton's tyrosine kinase. Oncogene 151375,1997 42. Li T, Tsukada S, Satterthwaite A, et a1 Activation of Bruton's tyrosine kinase f3TK) by a point mutation in its pleckstrin homology (PH) domain. Immunity 2451,1995 43. Maas A, Dingjan GM, Savelkoul HE, et al: The X-linked immunodeficiency defect in the mouse is corrected by expression of human Bruton's tyrosine kinase from a yeast artificial chromosome transgene. Eur J Immunol272180,1997 44. Mano H, Yamashita Y, Sat0 K, et al: Tec protein-tyrosinekinase is involved in interleukin-3 signaling pathway. Blood 85343, 1995 45. McKinney-RE J, Katz SL, Wilfert CM Chronic enteroviral m e ~ g ~ c e p h a ~int i s agammaglobulin~cpatients. Rev Infect Dis 9334,1987 46. Minegishi Y, Coustan SE, Rapalus L, et al: Mutations in Igu result in a complete block in 8-cell development at the pre-B cell receptor checkpoint. J Clin Invest 104:1115,1999
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47. Minegishi Y, Coustan SE, Wang YH, et al: Mutations in the human X5/14.1 gene result in B-cell deficiency and agammaglobulinemia. J Exp Med 18771,1998 48. Minegishi Y, Rohrer J, Coustan-Smith E, et ak An essential role for BLNK in human B-cell development. Science 2861954,1999 49. Nonoyama S, Tsukada S, Yamadori, T et al: Functional analysis of peripheral blood B cells in patients with X-linked agammaglobulinemia. J Immunol 161:3925,1998 50. Novina CD, Kumar S, Bajpai U, et a1 Regulation of nuclear localization and transcriptional activity of TFII-I by Bruton’s tyrosine kinase. Mol Cell Biol 195014,1999 51. Oeltjen JC, Malley TM, Muwty DM, et al: Large-scale comparative sequence analysis of the human and murine Bruton’s tyrosine kinase loci reveals conserved regulatory domains. Genome Res 7315,1997 52. Ohta Y,Haire RN, Litman RT, et ak Genomic organization and structure of Bruton’s agammaglobulinemia tyrosine kinase: Localization of mutations associated with varied clinical presentations and course in X chromosome-linked agammaglobulinemia. k o c Natl Acad Sci U S A 91:9062,1994 53. Park H, Wahl MI, Afar DE, et al. Regulation of Btk function by a major autophosphorylation site within the SH3 domain. Immunity 4515,1996 54. Pevear DC, Tull TM, Seipel ME, et al: Activity of pleconaril against enteroviruses. Antimicrob Agents Chemother 02109,1999 55. Rawlings DJ, Saffran DC, Tsukada, S et al: Mutation of unique region of Bruton’s tyrosine kinase in immunodeficient xid mice. Science 261:358,1993 56. Rawlings DJ, Scharenberg AM, Park H, et al: Activation of BTK by a phosphorylation mechanism initiated by Src family kinases. Science 271:822,1996 57. Resh MD: Myristylation and pahitylation of Src family members: The fats of the matter. Cell 76411,1994 58. Rohrer J, Conley ME Correction of X-linked immunodeficient mice by competitive reconstitutionwith limiting numbers of normal bone ma?row cells. Blood 94:3358,1999 59. Rohrer J, Minegishi Y, Richter D, et al: Unusual mutations in BTR An insertion, a duplication, an inversion, and four large deletions. Clin Immunol9028, 1999 et al: The genomic structure of human BTK, the 60. Rohrer J, Parolini 0, Belmont JW, defective gene in X-linked agammaglobulinemia. Immunogenetics 40319,1994 61. Roifman CM,Rao CP, Lederman HM, et al: Increased susceptibility to Mycoplasma infection in patients with hypogammaglobulinemia. Am J Med 80:590,1986 62. Saffran DC, Parolini 0, Fitch HM,et ak Brief report: A point mutation in the SH2 domain of Bruton‘s tyrosine kinase in atypical X-linked agammaglobulinemia. N Engl J Med 3301488, 1994 63. Scharenberg AM, El-Hillal 0, Fruman DA, et al: Phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3)/Teckinase-dependent calcium signaling pathway: A target for SHIP-mediated inhibitory signals. EMBO J 171961, 1998 64. Schiff GM, Sherwood J R Clinical activity of pleconaril in an experimentally induced coxsackievirus A21 respiratory infection. J Infect Dis 181:20,2000 65. Schmugge M, Lauener R, Seger RA, et a1 Chronic entemviral meningo-encephalitis in X-linked agammaglobulinaemia: Favourable response to anti-enteroviral treatment. Eur J Pediatr 1581010,1999 66. Sideras P, Muller S, Shiels H, et a1 Genomic organization of mouse and human Bruton’s agammaglobulinemia tyrosine kinase (Btk) loci. J Immunol 1535607, 1994 67. Smith CI, Baskin B, Humire GP, et al: Expression of Bruton’s agammaglobulinemia tyrosine kinase gene, BTK, is selectively down-regulated in T lymphocytesand plasma cells. J Immunol 152:557, 1994 68. Smith CIE, Witte ON X-Linked agammaglobulinemia: A disease of Btk tyrosine kinase. In Ochs HD, Smith CI, Puck JM (eds): Primary Immunodeficiency Diseases (A Molecular and Genetic Approach), ed 1. Oxford University Press, 1998, p 263 69. Stiehm E R Human intravenous immunoglobulin in primary and secondary antibody deficiencies. Pediatr Infect Dis J 16696,1997 70. Stuckey M, Quinn PA, Gelfand Ew: Identification of UreapZa$m urealyticum (T-strain Mycoplasma) in patient with polyarthritis. Lancet 2:917,1978 71. Takata M, Kurosaki T: A role for Bruton’s tyrosine kinase in B-cell antigen receptormediated activation of phospholipase C-y2. J Exp Med 184:31,1996
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72. Tamagnone L, Lahtinen I, Mustonen T, et ak BMX, a novel nonreceptor tyrosine kinase gene of the BTK/ITK/TEC/TXK family located in chromosome Xp22.2. Oncogene 93683,1994 73. Thomas JD, Sideras P, Smith CI, et a1 Colocalization of X-linked agammaglobulinemia and X-linked immunodeficiency genes. Science 261:355,1993 74. Touhara K, Inglese J, Pitcher JA, et a1 Binding of G protein p gamma-subunits to pleckstrin homology domains. J Biol Chem 269:10217,1994 75. Tsukada S, Saffran DC,Rawlings DJ, et a1 Deficient expression of a B-cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia. Cell 72279, 1993 Weening RS, Schellekens I T,et ak Colorectal cancer in patients with 76. van-der-Meer JW, X-linked agammaglobulinaemia. Lancet 341:1439,1993 77. Vetie D, Vorechovsky I, Sideras P, et ak The gene involved in X-linked agammaglobulinaemia is a member of the Src family of protein-tyrosinekinases. Nature 361:226,1993 78. Vihinen M, Belohradsky BH, Haire RN, et al: BTKbase, mutation database for X-linked agammaglobulinemia (XLA). Nucleic Acids Res 25366,1997 79. Vihinen M, Brandau 0, Branden LJ, et a1 BTKbase, mutation database for X-linked agammaglobulinemia (XLA). Nucleic.Acids.Res. 26:242, 1998 80. Vihinen M, Iwata T, h o n C, et a1 BTKbase, mutation database for X-linked agammaglobulinemia (XLA). Nucleic Acids Res 24160, 1996 81. Vihinen M, Kwan SP, Lester T, et al: Mutations of the human BTK gene coding for bruton tyrosine kinase in X-linked agammaglobulinemia. Human Mutat 13:280,1999 82. Wahl MI, Fluckiger AC, Kato RM, et ak Phosphorylation of two regulatory tyrosine residues in the activation of Bruton’s tyrosine kinase via alternative receptors. Proc Natl Acad Sci U S A 9411526,1997 83. Webster AD, Rotbart HA, Warner T, et a1 Diagnosis of enterovirus brain disease in hypogammaglobulinemic patients by polymerase chain reaction. Clin Infect Dis 17657,1993 84. Webster AD, Taylor RD, Furr PM, et al: Mycoplasmal (ureaplasma) septic arthritis in hypogammaglobulinaemia. Br Med J 1:478,1978 85. Wicker LS, M e r I X-linked immune deficiency (xid) of CBA/N mice. Curr Top Microbiol Immunol 12487,1986 86. Xu S, Tan JE, Wong EP, et a1 B-cell development and activation defects resulting in xid-like immunodeficiency in BLNK/SLP-65-deficient mice. Int Immunol 12397,2000 87. Yamada N, Kawakami Y, Kimura H, et a1 Structure and expression of novel proteintyrosine kinases, Emb and Emt, in hematopoietic cells. Biochem Biophys Res Commun 192:231, 1993 88. Yang W, Desiderio S BAP-135, a target for Bruton’s tyrosine kinase in response to B-cell receptor engagement. Proc Natl Acad Sci U S A 94604, 1997 89. Ye1 L, Minegishi Y, Coustan SE, et a1 Mutations in the )L heavy-chain gene in patients with agammaglobulinemia. N Engl J Med 3353486,1996
Address reprint requests to Hubert B. Gaspar, MD, PhD Molecular Immunology Unit Institute of Child Health 30 Guilford Street London WCIN IEH United Kingdom e-mail: [email protected]
0889-8561/01 $15.00
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X-LINKED AGAMMAGLOBULINEMIA Hubert 8. Gaspar, MD, PhD, and Christine Kinnon, BSc, PhD
B-lymphocyte development is essential for production of functional antibody and humoral immune responses. The mechanisms that regulate this process are complex and tightly regulated. Defects in nonredundant components of this pathway lead to a clinical and immunologic syndrome of congenital agammaglobulinemia with absence of B-cell development. X-linked aga~aglobulinemia(XLA) was the first of these conditions to be described and is the most common cause of congenital agammaglobulinemia. In 1993, abnormalities in the Bruton’s tyrosine kinase (BTK) gene were shown to be the cause of XLA. A considerable amount of research has since been dedicated to understanding the genetic basis and molecular pathogenesis of the disease.
HSTORY XLA was the first human immunodeficiency described for which the underlying defect was clearly identifed. In 1952 Bruton reported the case of an 8-year-old boy who had suffered from recurrent infections, including sepsis caused by pneumococcus of different serotypes. Subsequent analysis of serum by protein electrophoresis revealed a lack of gammaglobulin? Realizing the association between the absence of gammaglobulin and recurrent infections, Bruton started gammaglobulin replacement therapy and demonstrated considerable clinical improvement. Although there was no family history of other affected males in this
From the Moiecular Immunology Unit, Institute of Child Health, University College London, London, United Kingdom IMMUNOLOGY AND ALLERGY CLIMCS OF NORTH AMERICA
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VOLUME 21 NUMBER 1 FEBRUARY 2001
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initial case, subsequent studies revealed a similar clinical phenotype with an X-linked pedigree.=,35 The term agammaglobulinemia, first used by Bruton, continues to be associated with the condition. Although the phenotype of XLA is characterized in its classical form by an almost complete absence of immunoglobulin of all isotypes and the profound reduction of B lymphocytes in the peripheral circulation. Subsequent studies have shown that in some individuals sigruficant amounts of immunoglobulin are detectable in the peripheral blood.5,49 CLINICAL MANIFESTATIONS
The majority of affected boys are prone to recurrent bacterial infection with the peak incidence of onset at 6 months of age following the disappearance of maternal immunoglobulin.4oA study of 96 North American patients found that symptoms appeared in 25% of the patients by the age of 4 months, 50%by 8 months, and 75%by 12 months.4oIn a British study of 44 patients, 40% of patients presented in the first year of life and 21% remained asymptomatic until 3 to 5 years of life.3I In these two studies and another large retrospective the presenting features at diagnosis were variable but the most common clinical problems were pneumonia, otitis media, and gastrointestinal infection (Table 1). Other major infectious problems included arthritis, septicaemia, and meningitis. SPECTRUM OF INFECTIONS
Infections in X U usually are caused by pyogenic bacteria with ~ u e m o p ~ ~ infmzae, lus Staphylococcus aureus, Streptococcus pneumoniae, and Pseudomonas being the most common speciesa although many other types also have been reported.61,70,84 These reports were published before Table 1. INFECTIVE PRESENTATIONS IN PATIENTS WITH X U Numbers (%) of patients with X U in each study Presenting Infection Ear, nose, and throat infection Pneumonia Gastrointestinal infectiOnS Meningitis Septic arthritis Bacterial skin sepsis
Ledefman and WlnkeIsteiP
(n = 96) 72 (75) 54 (56) 34 (35) 10 (10) 8 (8) 27 (28)
X-LINKED AGAMMAGLOBULINEMLA
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the introduction of the H. influenme B vaccination programs, and although no recent studies have been published, it is likely that the spectrum of infection is now considerably different. Susceptibility to M y c ~ infections ~ ~ ~ remains s ~ ~ a problem in XLA and may cause a generalized systemic infection or be localized to the respiratory tract, joints, or urogenital tract. It is important to note that these infections may be of a more prolonged nature with an insidious onset. Arthritis
Arthritis is a common feature in XLA occurring in approximately 20% of patientsJ6S4OAlthough septic arthritis caused by pyogenic organisms is described, other patients suffer from a nonseptic monoarthritic form that characteristically affects the large joints, resulting in hydrarthrosis and a limited range of movement. There is usually no joint pain or evidence of joint destruction, and there is resolution of symptoms when ~ u n o g l o b therapy u~ is initiated or increased, suggesting an unidentified infectious cause. In addition to pyogenic bacterial and nonseptic forms of arthritis, enterovirus and mycoplasma species have also been identified in synovial fluid from other patients. Chronic infections
Lederman and WinkelsteinM also reported a high incidence of chronic infective complications with chronic pulmonary disease affecting approximately 50% of patients with XLA. The prevalence of pulmonary disease was age related, with 13% of affected patients under 10 years old, 41% between 10 and 20 years old, and 76% greater than 20 years of age. The other major chronic complications were hearing loss (32% of cases) caused by chronic otitis media and meningo-encephalitis and delayed speech acquisition (14% of cases). Enterovirai infections
Resistance to viral infection remains predominantly intact except for a susceptibility to enteroviral infection.45In two major retrospective analyses,3’rrnenteroviral infections were shown to cause a meningoencephalitis sometimes in association with a dermatomyositis-like syndrome. These infections were seen despite gammaglobulin replacement therapy in some individuals. The most. common organism isolated was echovirus, although coxsackie viruses A and B also have been reported to cause disease.45The incidence of enteroviral disease in current populations of XLA patients appears to be less frequent, a finding that may be as a result of improved clinical monitoring of patients and optimal immunoglobulin substitution therapy, This acute sensitivity of patients
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with XLA and other patients with hypogamrnmaglobulinemia to CNS enteroviral infection highlights the need for a thorough investigation of patients with XLA presenting with neurologic symptoms. The pattern of presentation can be variable, with some patients developing sIow progressive neurologic abnormalities including ataxia, paresis, loss of cognitive function, emotional lability, and personality changes. In others the onset is more acute and manifest by fever, headache, and seizures. Examination of the cerebrospinal fluid (CSF) shows a pleocytosis in most cases with a predominance of lymphocytes and a protein concentration typically between 0.5 and 5 g/L.& Culture of enterovirus has historically been difficult, but with the development of polymerase chain reaction (PCR) technologs identification of the causative agent has improved con~iderably.8~ Other Infections
As would be expected from a pure humoral abnormality, cell-mediated immunity remains predominantly intact. Increased frequency and severity of infection caused by nonbacte~alorganisms (other than enterovirus) is not a feature of XLA. Although these infections do occur, they are no more serious than in normal hdividuals and are selflimiting. Exceptions to this include a number of case reports of Pneumocystis carinii pneumonitis in patients with XLA and other hypogammaglobulinemic patients with intact T-cell function.' This suggests that despite the presence of anti-Pnarrnocystis antibodies in pooled immunoglobulin preparations, there may be a susceptibility to Pneumoqsfis infection. XLAANDCANCER
Although certain primary immunodeficiencies can predispose to an increased incidence of tumors (ataxia-telangiectasia, Wiskott-Aldrich syndrome, X-linked lymphoproliferative syndrome), such a relationship is not obvious for XLA. Lederman and Winkelstein documented two patients in their study? one with a B-cell lymphoma of the terminal ileum and the other with a reticulum cell sarcoma of the bowel. One patient with pituitary adenoma was reported by Hermaszewski and Web~ter.~' Analysis of a primary disease registry showed that of 500 patients with neoplasia, 4.2% were reported to have In a study of 52 Dutch patients with XLA, three unrelated young adults were found to have a rapidly progressive colorectal cancer, suggesting that patients with XLA have a 30-fold increased risk of d i e a ~ e This . ~ ~ finding has prompted some authors to propose that complete gastrointestinalstudies should be performed on all XLA patients. Most clinicians, however, reserve such thorough evaluation for those with documented atrophic gastritis where there is a higher risk of gastric cancer.
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MORTALITY
Smith and Witte@ analyzed three major retrospective studies of patients with XLA and found 30 deaths recorded among 170 patients (17.6%). The major cause of death was viral infection and was predominantly caused by disseminated echoviral infection. The other major cause of death was cardiorespiratory failure from chronic pulmonary disease and cor pulmonale. Other causes of death included amyloidosis, septicemia with osteomyelitis, and inflammatory bowel disease. These data are based on analysis of patients who were diagnosed and treated, in many cases, over 30 years ago and most likely do not reflect the current history of the disease. Now that an unambiguous molecular diagnosis can be made, and with improved awareness, antibiotic treatment, monitoring and treatment of the condition, the outlook for individuals with XLA should be much improved. HETEROGENEITY
Identification of BTK as the gene defective in XLA75,77 allows unambiguous assignment of a molecular defect to individuals with abnormalities in antibody production. Although the majority of BTK-deficient patients display the classical immunophenotype of less than 1%peripheral B lymphocytes and virtual absence of all immunoglobulin isotypes, a significant number of atypical or “leaky” phenotypes have been identified, implying that XLA as a disease entity has considerable clinical and immunologic heterogeneity. As many as 20% of patients with mutations in BTK have delayed onset of symptoms or higher concentrations of serum immunoglobulins than expected.” 28, 29, 38, 62 There also appear to be differences in clinical severity in some individuals who have very low concentrations of serum immunoglobulins and almost no B cells but surprisingly few infective 39 The reasons for clinical and immunologic heterogeneity in XLA are unclear and may be dependent on factors other than the BTK defect. IMMUNOPATHOLOGY
The major immunologic abnormality in XLA is the lack of B lymphocytes in the peripheral blood and other organs and the consequent absence of immunoglobulin production. Physical examination of patients with XLA shows only rudimentary adenoidal and tonsillar tissue and peripheral lymphoid hypoplasia. Histologic analysis of secondary lymphoid tissue demonstrates lack of germinal center and follicle formation.= In the lamina propria of the gut, plasma cells typically are absent. Examination of XLA bone marrow reveals a block in B-cell maturation that is variable with regard to the stage of B-cell maturation and severity of the developmentalarrest. In a study of eight patients, analysis
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GASPAR & KINNON
of bone marrow demonstrated a maturation arrest in the transition between early pre-B cells and pre-B cells? The severity of this block was variable with some patients, showing a normal number of pre-B cells in the bone marrow and suggesting that a later arrest in development was present in these individuals. In subsequent studies, it also has been shown that a differentiation block can occur earlier at the pro- to pre-Bcell transition,& although this block was not absolute (Fig. 1).Detailed analysis of peripheral blood from patients with XLA presenting classical phenotypes reveals that between 0.01% and 0.1% of CD19+ cells can still be detected.& Functional analysis of this B-cell population shows that these cells proliferate and produce i ~ u n o g l o b u l i nE (IgE) in response to CD40 and interleukin-4 (IL-4) stimulation, suggesting that cells that escape the developmental block are still capable of further maturati0n.4~ MOLECULAR BASIS OF XLA
The genetic defect in XLA was identified in 1993 by two groups, one using a positional cloning approachn and the other searching for novel protein kinases expressed in B lymphocyte^.^^ The gene was designated BTK and is located on the long arm of the X chromosome at Xq21.3. The human gene encompasses 37.5kb, is organized into 19 exens,=, 52, 66 including a 5’ untranslated region (exon l), and encodes a 659 amino acid protein. BTK expression is primarily restricted to hematopoietic cells and is found throughout B-cell differentiation from CD34’ pro-B cells to mature B cells but is down-regulated in antibody secreting plasma cells. Expression also is present in the monocytes, 67 There is, myeloid cells, erythrocyte precursors and megakaryocytes.22, however, no evidence of BTK expression in T-cell lineage or in natural killer (NK) cells. The Btk protein belongs to a family of cytoplasmic tyrosine kinases that are related to, but distinct from, the Src family kinases (Fig. 2). The protein is characterized by its modular structure that includes an aminoterminal pleckstrin homology (PH) domain followed by a tec-homology (TH) domain (which includes a proline rich region), an Src homology SH3 domain, an SH2 domain, and a carboxy-terminal kinase SH1 kinase domain. Catalytic activity resides in the kinase domain while the other domains are necessary for protein-protein interactions. Other members of this family include Tec,& Itk,” 87 and Bmx,” all of which are also expressed in hematopoietic tissues. Several key features distinguish the Btk/Tec family of proteins from the Src family kinases. Btk lacks the a ~ o - t e r m i n amyristylation l signal essential for post-translational modification and the membrane localization of S r ~ . At 5 ~ the a m i n ~ t e r m end ~ l of Btk is the PH domain that consists of seven antiparallel p sheets that form a putative ligand binding pocket on one face of the molecule. This pocket has been shown to bind specifically to phosphatidylinositols and phosphatidyl inositol-3,4,5-trisphosphate (PIP3)?4 Thus, in resting cells, Btk is localized in the cyto-
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GASPAR&KINNON
BtklTec family
src family
SywzAP-70 family
Figure 2. Btk is related to but distinct from the Src family of tyrosine kinases. It shares kinase, SH2, and SH3 domains but has a unique amino-terminal PH and TH domain.
plasm, and membrane recruitment of Btk is mediated by a Btk-PH domain and PIP, interaction. The C-terminal portion of the PH domain contains a highly conserved tryptophan residue, is folded into an ahelix, and can modulate binding to the p.v subunit of heterotrimeric G pr~teins.’~ Btk also lacks the carboxy-terminal tyrosine residue kinases (Y-527in Src) that negatively regulate the catalytic activity of Src kinases? Regulation of Btk catalytic activity is more complex, as demonstrated by the structurally homologous T-cell kinase Itk which shows an intramolecular association of the SH3 domain with the proline rich region of the TH domain thus restricting the access of the SH3 domain to potential Phosphorylation of a tyrosine residue within the SH3 domain may disrupt SH3-TH interaction thereby liberating SH3 and TH domains to form associations with alternative ligands. A summary of the interactions and functions of Btk is illustrated in Table 2. BTK FUNCTION AND ITS ROLE IN B-CELL DEVELOPMENT
Tyrosine kinases have been studied in many hematopoietic cell lineages and have been shown to act as signal transduction molecules mediating cell surface receptor activation events to downstream pathways. Cross-linking of a number of cell surface receptors, including interleukin-5 (IL-5), IL-6, CD38, FcRe, and most importantly surface IgM on B cells, results in the recruitment of cytosolic Btk to the plasma membrane and activation of Btk by tyrosine phosphorylation.41The process of Btk activation is initiated by phosphorylation of tyrosine 551 (Tyr551) in the kinase domain of Btk by an Src family kinase, most likely Lyn, followed by Btk autophosphorylation at tyrosine 223 (Tyr223) in
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GASPAR&KINNON
the SH3 domain.53*56*82 Btk is then recruited to the cell membrane, a process depends on an intact PH domain and its association with PIP3.16,= At the cell membrane, activated Btk interacts with phospholipase-y2 (PLC-y2) leading to enhanced tyrosine phosphorylation of PLC-y2 and resulting in accumulation of inositol 1,4,5-triphosphate (IP3) and release of calcium from internal stores. This pathway has been well defined in in vitro models13,63 and is supported by evidence from Btk-deficient cell lines that show reduced phosphorylation of PLC-y2 and decreased 71 The detailed molecular mechanisms through calcium which Btk interacts with PLC-y2 are being defined. An adapter molecule, the B-cell-linker protein (BLNK), has been identified that mediates Btk interactions with PLC-y2.15 Following B cell receptor (BCR) crosslinking, activated Syk phosphorylates BLNK on a number of tyrosine residues.15 BLNK is then able to bind through phosphorylated tyrosine motifs to the PLC-y S H 2 domains15 and also to the S H 2 domain of Btk.27 Thus, BLNK may act to bring Btk in close proximity with PLC-y2 and allow Btk phosphorylation of PLC-y2 (Fig. 3). This is not an absolute mechanism because Btk is able to bind to unphosphorylated BLNK and PLC-y2 phosphorylation can occur in the absence of BLNK.2' Optimal function is only seen if phosphorylated BLNK is present, however. The crucial role of BLNK in B-cell development is further illustrated by the phenotypes of BLNK human and murine mutants that show distinct similarity to the respective Btk mutant phenotypes.48, Other biochemical consequences of Btk activation include coimmunoprecipitation with and phosphorylation of a protein termed Btk-associated protein-135 (BAP-135),= which has been independently identified as a DNA-binding protein TFII and thus links Btk with regulation of transcriptional e~ents.2~.Despite the large volume of biochemical and signaling data available, a relationship with cellular events and specifically the mechanism of B-cell development arrest has yet to be established. Clearly, Btk participates in a signal cascade responsible for the mobilization of calcium, but this can initiate several types of transcriptional events and growth responses such as proliferation and apoptosis. The close homology between Btk and the Src family kinases initially suggested that dysregulated Btk may have oncogenic potential. This finding was supported by random retroviral-mediated mutagenesis studies which identified a specific PH mutation that can lead to mutant Btk with enhanced transforming ability." Expression of the E41K mutant (termed Btk*) in NIH3T3 cells resulted in growth of cells in soft agar and was accompanied by an increase in membrane association and tyrosine phosphorylation at Y551 and Y223, suggesting increased activation. A mutation within the SH3 domain potentiated the transforming potential of Btk,* supporting the idea that the SH3 domain has a negative regulatory function. MUTATION ANALYSIS IN XLA
Identification of the BTK gene has led to research in mutational analysis by a number of groups worldwide. Over 500 unique mutations
34
GASPAR & KINNON
in BTK have been identified, and an international database of mutations and clinical i n f o ~ a t i o nhas been established (BTKbase web page: http:/ /www.uta.fi/laitokset/imt/bi~info/BTKbase).~~~ 79, Various different types of genetic a b n o ~ ~ t ihave e s been found in the BTK gene. One third of mutations are missense mutations, and these have been found p r e d o m i n ~ ~ inythe kinase domain of the gene, although they have been found in all domains except the SH3 domain.s0, Premature stop codons, deletions, and insertions also have been described throughout the gene. Although the large majority of mutations are found in the coding region, 12% of mutations have been found to affect splice site recognition sequence^.^ Only one mutation has been found in the promoter region, and this mutation is thought to affect transcriptional regulation of the About 5% to 10% of mutations result in gross alterations of the BTK gene.59In addition to large deletions, an inversion, a duplication, and a retroviral insertion have been identified. Despite the large quantity of mutation and phenotypic data available, it has not been possible to make definitive genotypephenotype correlations. The strongest argument for this comes from reports of considerable variation in clinical severity even within the same pedigree13,39suggesting that factors other than the genetic mutation may influence the clinical course. It has been suggested that, as in other i~nmunodeficiencies,’~ the presence of cytokine gene polymorphisms or defects in innate immunity caused by mutations in m ~ a n - b i n d i n g lectin may be important. There is, however, no evidence to refute or confirm these ideas. It also is possible that environmental factors may play an important role. The consequence of the gene mutation on expression of the Btk protein and its relation to clinical severity also has been explored. It has been found that in the majority of individuals, despite the severity of the clinical phenotype and regardless of the nature of the mutation, there is a complete lack of Btk expression.17,22, 29 It would appear that even c ~ b o x y - t e ~ i namino al acid substitutions render the messenger RNA (mRNA) or Btk protein unstable. A N I ~ A LMODELS
The consequence of mutations in BTK also have been studied in murine models of the disease. The X-linked immunodeficiency (xid) mouse has a single amino acid substitution (R28C) in the PH domain of Btk.55,73 The immunophenotype of the xid mouse shows normal serum concentrations of IgG1, IgG2a and IgGZb but markedly reduced 1 6 3 and IgM concentrations. Mice also are unable to make antibody responses to T-cell-independent antigens but respond well to stimulation with T-cell-dependent antigens.85The block in B-cell maturation is much less severe in xid mice compared with patients with XLA. Whereas typical patients with XLA have less than 1% of the normal number of peripheral B cells, B-cell numbers in xid mice are only reduced to 30% to 50% of the normal amount. In both species, defects in BTK are associated with an immature B-cell phenotype with the majority of B
X-LINKED AGAMMAGLOBULLNEMIA
35
cells dispaying an increased density of surface IgM.” 85 The differences between the murine and human phenotypes cannot be explained by the nature of the xid genetic defect because human R28C mutations with severe phenotypes have been de~cribed.~ Furthermore, Btk-null mice created by homologous recombination also have a B-cell phenotype that 36,37 is identical to the xid DIAGNOSIS
The diagnostic criteria for XLA agreed on by members of the PanAmerican Group for Immunodeficiency (PAGTT))and the European Society for ImmundeficiencyS (ESID) are listed as follows: Definitive Male patient with CD19+ B cells less than 2% and at least one of the following: Mutation in BTK Absent BTK mRNA or protein in peripheral blood mononuclear cells or neutrophils Maternal family history of agammaglobulinemiaand abnormal Bcell numbers Probable Male patient with CD19+ B cells less than 2% in whom all of the following are positive: Onset of recurrent bacterial infections in the first 5 years of life Serum IgG, IgM, and IgA more than 2 standard deviations below normal for age Absent isohemagglutinins or poor vaccine responses Other causes of hypogammaglobulinemia have been excluded Possible Male patient with CD19+ B cells less than 2% in whom other causes of hypogammaglobulinemiahave been excluded and at least one of the following is positive: Onset of recurrent bacterial infections in the first 5 years of life %nun IgG, IgM and IgA more than 2SD below normal for age Absent isohemagglutinins or poor vaccine responses Atypical presentations 15% to 20% of patients have higher concentrations of serum immunoglobulins than expected or are not recognized until after 5 years of age Differential diagnosis p heavy chain deficiency A5 surrogate light chain deficiency Iga deficiency BLNK deficiency
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GASPAR & KINNON
A pattern of X-linked inheritance, severely decreased numbers of peripheral B lymphocytes, and decreased immunoglobulin production makes a clinical diagnosis of XLA very likely. Because approximately one third to one half of XLA cases are sporadic and with the identification of genetic defects are giving rise to autosomal recessive forms of the condition, alternative mechanisms of diagnosis are necessary. There is considerable heterogeneity to the XLA phenotype, as previously discussed, making a diagnosis based on clinical and immunologic criteria difficult. Identification of BTK as the defective gene in XLA allows mutation analysis to be performed, can offer an unambiguous molecular diagnosis, and offers possibilities for carrier assignment and prenatal diagnosis. Mutation analysis for XLA normally involves screening the 19 exons of the BTK gene in genomic DNA by one of many techniques of which single-stranded confirmational polymorphism (SSCP) analysis is the most commonly used before direct sequencing of the affected exon. SSCP analysis can be a time consuming and expensive procedure, and a definitive diagnosis may take weeks to achieve. Furthermore, some mutations may not be detected by SSCP because the sensitivity of the technique is only 85% to 90%. Other screening techniques do exist, but the sensitivities are not necessarily greater than that of SSCP. Examination of Btk protein expression in peripheral blood monocytes by either Western blot or flow cytometric analysis offers a more rapid and equally sensitive diagnostic assay. In flow cytometry studies, 40 of 41 patients were shown to lack Btk expression,17and the diagnosis was made in 10 individuals for whom no previous genetic data was available. In the authors' own experience, 29 of 31 patients had abnormal expression by Western blot analysis with the majority of patients completely lacking expression and two cases showing the presence of abnormally si~ed-protein'~ (and unpublished data). Thus, over 90% of patients with XLA have abnormalities in Btk expression. In the authors' immunodeficiency diagnostic service, the routine strategy is to screen initially by immunoblotting and then refer abnormal findings for mutation analysis. Thus, a rapid diagnosis can be established, and SSCP analysis can be undertaken with greater confidence. If Btk expression is normal, clinical and immunologic findings dictate when referral for genetic testing is appropriate. Immunoblot expression assays are not routinely of use in determining carrier status because of the absence of protein in most patients; however, in one patient in whom a truncated protein was found caused by a frameshift mutation, immunoblot analysis of cells from the mother showed the presence of an abnormal and normal band, confirming her carrier status (unpublished data). The flow cytometric analysis assay does offer the possibility of carrier testing as monocytes from obligate carriers of patients with no Btk expression show a dual population of Btk-expressing and nonexpressing cells.17 The differential diagnosis of XLA, especially in those patients lacking an X-linked pedigree, has increased significantly. Congential agammaglobulinemia with absent B cells has been shown to arise as a result A 5 surrogate of defects in at least four other genes (i.e., p heavy ~hain,8~
X-LINKED AGAMMAGLOBULINEMIA
37
light chain:7 Iga& BLNK&)C, all of which are essential to ~ c t i o n i n g through the B- or pre-B-cell receptor complex. Males with these defects are clinically indistinguishable from those with XLA, although in p and IgoL chain deficiency., examination of B-cell precursors in the bone marrow shows there is a more stringent block in B-cell development at the pro- to pre-&ell transition.&It must be remembered, however, that these autosomal defects are very rare. In a recent analysis of a large chort of males with presumed XLA, 90% to 95% had mutations in BTK? A rationale for diagnosis would be initially to undertake protein and genetic analysis for BTK defects. If no BTK defect was identified, bone marrow sample analysis by RT-PCR and flow cytometry for examining the expression of these candidate genes may prove useful in determining the specific defect. CLINICAL MANAGEMENT
The mainstay of treatment in XLA is immunglobulin replacement therapy. In most institutions, replacement is given on a regular basis through intravenous immunoglobulin (IVIG),but difficulty with venous access in young children and ease of administration has led to some centers adopting subcutaneous administration (SCIG) as an alternative approach. The use of IVIG replacement is now routine in most centers. Treatment aims to achieve serum IgG levels in the normal age-related range. For most patients, treatment involves a dose of approximately 0.4 to 0.6 g/kg every 3 to 4 weeks. Some centers practice a loading dose of 1 g/kg at the initiation of therapy. Dose and frequency should be tailored to the trough levels of immunoglobulin and clinical symptoms. In certain adult centers the practice is to keep levels above 8 g/L for individuals who continue to have recurrent infective problems. This practice may be especially important for the prevention of chronic lung disease. Once established on a regular regime, patients can be trained to self-cannulate and administer M G at home, leading to greater patient convenience. Adverse reactions can be associated with the infusion of IVIG, most commonly including headaches, nausea, vomiting, flushing, and m~algia.6~ More severe symptoms, such as chest pain and anaphylactoid reactions, have been described but are rare. Thus, IVIG should only be given under the supervision of experienced practitioners, and patients on home therapy should be taught to recognize and manage such events. Rapid subcutaneous infusion of immunoglobulin is an alternative o n has many advantages. First used in adult method of a ~ i s ~ a t i that patients with common variable immunodeficiency it is useful in the pediatric population in whom regular venous access can be a difficult and traumatic process. SCIG allows ease of access and is more readily transferable to home a d ~ t r a t i o n Early . studies also suggest that there is no difference in the maintenance of optimal immunoglobulin levelsz0;however, only a limited volume of immunoglobulin can be administered in one infusion, increasing the frequency of infusions,
38
GASPAR&KINNON
especially in older children. Local adverse reactions also have been observed in a few children. Only a concentrated intramuscular preparation, which has not undergone the virus inactivation steps of IV preparations, has been available for use to date, A trial of a specific virus inactivated subcutaneouspreparation currently is underway and demonstration of efficacy with this product may lead to increasing the use of this mode of immunoglobulin administration. Despite the excellent clinical response of most patients to immunoglobulin replacement therapy, it is still important to monitor patients on a regular basis. It is recommended that trough immunoglobulin levels are monitored regularly to ensure that optimal levels are maintained. Liver function tests also should be checked regularly so that any hepatic illness caused by viral transmission can be detected at an early stage. In the authors' practice, all children undergo a chest radiograph or chest CT scan on a yearly basis, as nonsymptomatic infections can occur and can lead to chronic changes. Early detection of such abnormalities can prevent further progression by optimizing replacement therapy or by use of prophylactic antibiotics. Enteroviral disease and especially chronic mening~nc~halitis remain the greatest dangers to patients with XLA and other agammaglobulinemic patients. Effective antiviral agents ha3e not been previously available, but recent reports suggest that pleconaril, an orally active broad-spectrum antipicomaviral agent with excellent penetration of the central nervous system and nasal epithelium, may have significant antienteroviral activity. Studies show that pleconaril has sigruficant activity against the common forms of enteroviral strains in in vitro and in vivo murine models." In an experimentally induced human model of coxsackie A21 respiratory infection, prophylactic use of pleconaril resulted in significant reductions in viral shedding in nasal secretions, nasal mucus production, and total respiratory illness score^.^ One case report has documented its efficacy in treating enteroviral meningoencephalitis by reduction of CSF inflammatory markers and resolution of clinical signs and symptoms.* Its use currently is being piloted in immunodeficient patients, but published results are not yet available. GENE THERAPY
Gene therapy for XLA appears to be an attractive concept as only the B-cell lineage needs to be targeted. Because female carriers show nonrandom X-inactivation in B cells? l1 it would appear that there would be a survival advantage for corrected cells. Transgenic mice experiments have demonstrated the principle that BTK gene expression can overcome the B-cell developmental defects seen in BTK null mice.lO, In one strategy, a yeast artificial chromosome (YAC) transgenic mouse strain was generated in which high level expression of human BTK was provided by endogenous regulatory cis-acting elements present on a 340-kb transgene. When the YAC-btk transgenic mice were mated with the
X-LINKED AGAMMAGLOBULINEMIA
39
BTK- strain,3O BTK- mice with a YAC-btk background were generated. Analysis of the immunophenotype in this population demonstrated restoration of normal numbers of mature B cells in the bone marrow and periphery and reconstitution of the CD5+ B-cell population in the peritoneum. The expression of the YAC-btk transgene restored serum Ig levels and responses to T-cell independent antigens in BTK- mice. In an alternative strategy, a transgenic mouse strain was generated in which expression of the human BTK gene (complimentary DNA [cDNA]) was placed under the control of the murine class 11 major histocompatibility complex Ea gene locus control region, which provides gene expression from the pre-B cell stage onwards.l0 Again, when these mice were mated with the btk- strain, the presence of the transgene on the BTKbackground resulted in the rescue of the btk- strain immune deficits. The success of this latter experiment suggests that the BTK gene does not necessarily need to be introduced into hematopoietic stem cells or pro-B cell populations to restore B-cell development, and tight regulation of BTK expression is not necessary for successful reconstitution. In more recent competitive reconstitution experiments, xid mice defects were corrected when sublethally irradiated mice received a mixture of 25,000 normal cells and 475,000 xid cells.%The identity of cells responsible for reconstitution in this study was shown to be early hematopoietic presursors (CD19- B220 - ) that had not yet committed to the B-cell lineage. Reconstitution was achieved with as little as 10% of normal cells, suggesting that relatively inefficient gene transfer may be successful because of the strong survival advantage for normal cells. SUMMARY
X-linked agammaglobulinemia remains the prototypic humoral immunodeficiency with absence of immunoglobulin production caused by an abnormality in B-cell development. Identification of BTK as the defective gene has led to a greater understanding of the molecular mechanisms involved in signaling downstream of B-cell surface receptors, although the pathogenesis of the arrest in B-cell development remains to be determined. Identification of BTK also has led to improved methods of diagnosis and carrier detection and has demonstrated the clinical heterogeneity of BTK abnormalities. This raises further questions regarding the factors that ultimately determine the severity of the clinical phenotype. Clinical management of XLA has been dependent on gammaglobulin substitution, and a variety of preparations and modes of administration are now available. The outlook for patients with XLA is good as long as patients are carefully monitored and maintained on adequate immunoglobulin substitution. With the development of effective antienteroviral therapy, the major cause of mortality in XLA may soon be treatable. Observations from carrier females and murine studies all suggest that XLA may be a good candidate for gene therapy. There is a strong selective advantage for corrected cells, and tight regulation
40
GASPAR&KI"ON
of gene transfer is not a necessity. Thus, new possibilities for treatment may soon become available.
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