Test ID: BTKMP
Bruton's Tyrosine Kinase (BTK) Genotype and Protein Analysis, Known Mutation Sequencing and Flow Cytometry

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 both protein and gene analysis, this test provides a comprehensive assessment and enables appropriate genotype-phenotype correlations

When this test is ordered, a DNA extraction will always be performed at an additional charge.

• Informed Consent for Genetic Testing
• Bruton's Tyrosine Kinase (BTK) Genotype Patient Information Sheet

BTKKM/29304, BTKKQ/89286: Polymerase Chain Reaction (PCR) followed by DNA Sequence Analysis

(PCR is utilized pursuant to a license agreement with Roche Molecular Systems, Inc.)

BTK/89011: Flow Cytometry

Forms:

1. Bruton's Tyrosine Kinase (BTK) Genotype Patient Information Sheet (Supply T620) in Special Instructions

2. New York Clients-Informed consent is required. Please document on the request form or electronic order that a copy is on file. An Informed Consent for Genetic Testing (Supply T576) is available in Special Instructions.

Ordering physician name and phone number are required.

For serial monitoring, we recommend that specimen draws be performed at the same time of day.

Specimens must arrive within 72 hours of draw. Send specimens Sunday through Thursday only. Draw and package specimens as close to shipping time as possible. Ship specimens overnight.

Two separate EDTA specimens are required.

Specimen Type: Blood for BTKKM/29304 Bruton's Tyrosine Kinase (BTK) Genotype, Known Mutation and BTKKQ/89286 Bruton's Tyrosine Kinase (BTK) Genotype, Known Mutation Sequencing

Container/Tube: Lavender top (EDTA)

Specimen Volume: 3 mL

Collection Instructions:

1. Send specimen in original tube.

2. Label as BTKKM/29304 and BTKKQ/89286.

Additional Information: Transfusions will interfere with testing for up to 4 to 6 weeks. DNA obtained from white cells may not provide useful information for patients who received a recent transfusion of blood that was not leukocyte-reduced. Wait 4 to 6 weeks until transfused cells have left the patient's circulation before drawing the patient's blood specimen for genotype testing.

Specimen Type: Blood for BTK/89011 Bruton's Tyrosine Kinase (Btk), Protein Expression, Flow Cytometry, Blood
Container/Tube: Lavender top (EDTA)

Specimen Volume: 5 mL

Collection Instructions:

1. Send specimen in original tube. Do not aliquot.

2. Label as BTK/89011.

X-linked agammaglobulinemia (XLA) is a humoral primary immunodeficiency affecting males in approximately 1/200,000 live births. XLA is caused by mutations in the Bruton's 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 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 over-represented 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 has been reported in only 1 female patient (6) and, therefore, females with clinical features that are identical to XLA should be evaluated both for XLA (especially when there is a family history of XLA) and for autosomal recessive agammaglobulinemia.

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)

An interpretive report will be provided.

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.

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).

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, Edvard 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

Genomic DNA is first extracted from whole blood, followed by BTK gene amplification by PCR. Only the specific familial mutation will be evaluated in this genetic test. The PCR product is purified from unincorporated primers and nucleotides by enzymatic digestion and sequenced in both directions using sequencing primers and fluorescent dye-terminator chemistry. Sequencing products are separated on an automated sequencer and trace files will be analyzed for the known familial mutation in the appropriate exon(s) and/or intron/exon boundaries using specialized mutation detection software (Phredphrap/ Consed/ Polyphred) and visual inspection. (Unpublished Mayo method)

The Btk protein expression flow cytometry assay is carried out with a whole blood sample. The cells in the blood are stained with antihuman CD20 (B cells) and CD14 (monocytes) antibodies, followed by RBC lysis (using a premade Lysis buffer), cell fixation and permeabilization to prepare the cell membrane for the antihuman Btk antibody. After the permeabilization step, the cells are stained for intracellular Btk using an antihuman Btk-fluorescent preconjugated antibody from BD Biosciences. After the staining and wash process, the cells are resuspended in 500 uL of BD FACS stain buffer in the final step of the assay and the sample is analyzed by multiparametric flow cytometry. (Unpublished Mayo method; 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)

Varies

Bruton's Tyrosine Kinase (BTK) Genotype, Known Mutation Sequence

81479

Bruton's Tyrosine Kinase (Btk), Protein Expression, Flow Cytometry, Blood

88184