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Preferred test for confirming a diagnosis of X-linked agammaglobulinemia (XLA) in male family members of affected individuals with known BTK mutations
Preferred test for determining carrier status of female relatives of male XLA patients with known BTK mutations
By including protein and gene analysis, this test provides a comprehensive assessment and enables appropriate genotype-phenotype correlations.
X-linked agammaglobulinemia (XLA) is a humoral primary immunodeficiency affecting males in approximately 1 in 200,000 live births. XLA is caused by mutations in the Bruton tyrosine kinase gene (BTK),(1) which results in a profound block in B-cell development within the bone marrow and a significant reduction, or complete absence, of mature B cells in peripheral blood.(2) Approximately 85% of male patients with defects in early B-cell development have XLA.(3) Due to the lack of mature B cells, XLA patients have markedly reduced levels of all major classes of immunoglobulins in the serum and are, therefore, susceptible to severe and recurrent bacterial infections. Pneumonia, otitis media, enteritis, and recurrent sinopulmonary infections are among the key clinical diagnostic characteristics of the disease. The spectrum of infectious complications also includes enteroviral meningitis, septic arthritis, cellulitis, and empyema, among others. The disease typically manifests in male children younger than 1 year of age.
BTK, the only gene associated with XLA, maps to the X-chromosome at Xq21.3-Xq22 and consists of 19 exons spanning 37.5 kb genomic DNA.(4) BTK encodes a nonreceptor tyrosine kinase of the Btk/Tec family. The Bruton tyrosine kinase (Btk) protein consists of 5 structural domains (PH, TH, SH3, SH2, and TK). Mutations causing XLA have been found in all domains of the BTK gene, as well as noncoding regions of the gene. Missense mutations account for 40% of all mutations, while nonsense mutations account for 17%, deletions 20%, insertions 7%, and splice-site mutations 16%. Over 600 unique mutations in the BTK gene have been detected by full gene sequencing and are listed in BTKbase, a database for BTK mutations (http://bioinf.uta.fi/BTKbase).(5) Genotype-phenotype correlations have not been completely defined for BTK, but it is clear that nonsense mutations are overrepresented 4-fold compared to substitutions, which indicates that the latter may be tolerated without causing a phenotype. The type and location of the mutation in the gene clearly affects the severity of the clinical phenotype. Some mutations manifest within the first year or 2 of life, enabling an early diagnosis. Others present with milder phenotypes, resulting in diagnosis later in childhood or in adulthood.(5) Delayed diagnoses can be partly explained by the variable severity of XLA, even within families in which the same mutation is present. While the disease is considered fully penetrant, the clinical phenotype can vary considerably depending on the nature of the specific BTK mutation.(5) Lyonization of this gene is not typical and only 1 case of XLA in a female has been reported so far due to skewed lyonization in a carrier female. Therefore, females with clinical features that are identical to XLA should be evaluated for autosomal recessive agammaglobulinemia when deemed clinically appropriate(6) and for XLA, if a male parent is affected with the disease.
A flow cytometry test for intracellular Btk in monocytes using an anti-Btk monoclonal antibody was developed by Futatani et al, which was used to evaluate both XLA patients and carriers.(7) In this study, 41 unrelated XLA families were studied and deficient Btk protein expression was seen in 40 of these 41 patients, with complete Btk deficiency in 35 patients and partial Btk deficiency in 5 patients. One patient had a normal level of Btk protein expression. The 6 patients with partial or normal Btk expression had missense BTK mutations. Additionally, the flow cytometry assay detected carrier status in the mothers of 35 of the 41 patients (approximately 85%). In the 6 patients where the Btk expression was normal in the mothers of XLA patients, it was noted that all these patients were sporadic cases without previous family history of the disease.(7)
It appears, therefore, that most BTK mutations result in deficient expression of Btk protein, which can be detected by flow cytometry in monocytes.(7,8) Also, the mosaic expression of Btk protein in the monocytes by flow cytometry is potentially useful in the diagnosis of female carriers.(8) The flow cytometry test therefore provides a convenient screening tool for the diagnosis of XLA with confirmation of the diagnosis by BTK genotyping.(7,8) In the rare patient with the clinical features of XLA but normal Btk protein expression, BTK genotyping must be performed to determine the presence of a mutation.
A diagnosis of XLA should be suspected in males with 1) early-onset bacterial infections, 2) marked reduction in all classes of serum immunoglobulins, and 3) absent B cells (CD19+ cells). The decrease in numbers of peripheral B cells is a key feature, though this also can be seen in a small subset of patients with common variable immunodeficiency (CVID). As well, some BTK mutations can preserve small numbers of circulating B cells and, therefore, all the 3 criteria mentioned above need to be evaluated. Patients should be assessed for the presence of Btk protein by flow cytometry, although normal results by flow cytometry do not rule out the presence of a BTK mutation with aberrant protein function (despite normal protein expression). The diagnosis is established or confirmed only in those individuals who have a mutation identified in the BTK gene by gene sequencing or who have male family members with hypogammaglobulinemia with absent or low B cells. Appropriate clinical history is required with or without abnormal Btk protein results by flow cytometry.
It was shown that there are XLA patients with mothers who have normal Btk protein expression by flow cytometry and normal BTK genotyping and that the mutation in the patient is a result of de novo mutations in the maternal germline. In the same study, it was shown that there can be female carriers who have normal Btk protein expression but are genetically heterozygous, and they do not show abnormal protein expression due to extreme skewed inactivation of the mutant X chromosome.(6) Also, the presence of 1 copy of the normal BTK gene and associated normal Btk protein can stabilize mutant protein abrogating the typical bimodal pattern of protein expression seen in female carriers. Therefore, female carrier status can only conclusively be determined by genetic testing, especially if the Btk protein flow test is normal.
It is important to keep in mind that the mere presence of BTK gene mutations does not necessarily correlate with a diagnosis of XLA unless the appropriate clinical and immunological features are present.(9,10)
BTKKM: An interpretive report will be provided.
BTK: Bruton tyrosine kinase (Btk) expression will be reported as present, absent, partial deficiency, or mosaic (carrier).
A patient-specific interpretive report is provided.
This test should only be ordered if the familial BTK gene mutation is already known and the specific information is available to the laboratory.
Patients who have received a heterologous blood transfusion within the preceding 6 weeks, or who have received an allogeneic blood or marrow transplant, can have inaccurate genetic test results due to presence of donor DNA.
Rare polymorphisms could potentially lead to false-negative or false-positive results. If results obtained do not match clinical findings, additional testing should be considered. Any error in the diagnosis or in the pedigree provided to the laboratory could lead to an erroneous interpretation of results.
This method will not detect mutations that occur in intronic (other than exon-intron boundaries) and regulatory regions of the gene or large rearrangement-type mutations (which could cause a false-negative result).
Btk protein and genetic tests are not meant for patients with hematological neoplasias on kinase inhibitor therapy, including but not restricted to the selective Btk inhibitor, Ibrutonib. This test is only meant for the assessment of patients with a suspected monogenic primary immunodeficiency, X-linked agammaglobulinemia, caused by germline mutations in the Bruton tyrosine kinase gene.
1. Tsukada S, Saffran DC, Rawlings DJ, et al: Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia. Cell 1993;72:279-290
2. Noordzij JG, de Bruin-Versteeg S, Comans-Bitter WM, et al: Composition of precursor B-cell compartment in bone marrow from patients with X-linked agammaglobulinemia compared with health children. Pediatr Res 2002;2:159-168
3. Conley ME, Broides A, Hernandez-Trujillo V, et al: Genetic analysis of patients with defects in early B-cell development. Immunol Rev 2005;203:216-234
4. Lindvall JM, Blomberg KEM, Vargas L, et al: Bruton's tyrosine kinase: cell biology, sequence conservation, mutation spectrum, siRNA modifications, and expression profiling. Immunol Rev 2005;203:200-215
5. Valiaho J, Smith CI, Vihinen M: BTKbase: The mutation database for X-linked agammaglobulinemia. Hum Mutat 2006;27:1209-1217
6. Takada H, Kanegane H, Nomura A et al: Female agammaglobulinemia due to the Bruton's tyrosine kinase deficiency caused by extremely skewed X-chromosome inactivation. Blood 2004;103:185-187
7. Futatani T, Miyawaki T, Tsukada S, et al: Deficient expression of Bruton's tyrosine kinase in monocytes from X-linked agammaglobulinemia as evaluated by a flow cytometric analysis and its clinical application to carrier detection. Blood 1998;91(2):595-602
8. Kanegane H, Futatani T, Wang Y, et al: Clinical and mutational characteristics of X-linked agammaglobulinemia and its carrier identified by flow cytometric assessment combined with genetic analysis. J Allergy Clin Immunol 2001;108:1012-1020
9. Graziani S, Di Matteo G, Benini L et al: Identification of a BTK mutation in a dysgammaglobulinemia patient with reduced B cells: XLA or not? Clin Immunol 2008;128:322-328
10. Fleisher TA, Notarangelo LD: What does it take to call it a pathogenic mutation? Clin Immunol 2008;128:285-286