Test ID: BTKS
Bruton's Tyrosine Kinase (BTK) Genotype, Full Gene Sequence

Confirming a diagnosis of X-linked agammaglobulinemia (XLA) in male patients with a history of recurrent sinopulmonary infections, profound hypogammaglobulinemia, and <1% peripheral B cells, with or without abnormal Bruton’s tyrosine kinase (Btk) protein expression by flow cytometry

Follow-up testing when evaluating symptomatic male family members with an abnormal or equivocal result for Btk-protein expression by flow cytometry (BTK/89011 Bruton’s Tyrosine Kinase [Btk], Protein Expression, Flow Cytometry, Blood)

Evaluating for the presence of BTK mutations in female relatives (of male XLA patients) who do not demonstrate carrier phenotype by Btk flow cytometry

Because genotype-phenotype correlation is important for the diagnosis of XLA, the preferred test for confirming a diagnosis of XLA in males and identifying carrier females is BTKFP/89742 Bruton’s Tyrosine Kinase (BTK) Genotype and Protein Analysis, Full Gene Sequence and Flow Cytometry

• Informed Consent for Genetic Testing
• Bruton's Tyrosine Kinase (BTK) Genotype Patient Information Sheet
• Multiple Whole Blood EDTA Genotype Tests

Polymerase Chain Reaction (PCR) Followed by DNA Sequence Analysis

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

Multiple whole blood EDTA genotype tests can be performed on a single specimen after a single extraction. See Multiple Whole Blood EDTA Genotype Tests in Special Instructions for a list of tests that can be ordered together.

Container/Tube: Lavender top (EDTA)

Specimen Volume: 3 mL

Collection Instructions: Send specimen in original tube.

Additional Information:

1. Ordering physician name and phone number are required.

2. Bone marrow transplants will interfere with testing. Call Mayo Medical Laboratories at 800-533-1710 or 507-266-5700 for instructions.

3. 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.

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.

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 diagnostic clinical 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.

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 BTK 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 with 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 2 years 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 has been reported so far.  Therefore, females with clinical features that are identical to XLA should be evaluated for autosomal recessive agammaglobulinemia when deemed clinically appropriate.(6)

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 3 of the criteria mentioned above need to be evaluated.

The preferred approach for confirming a diagnosis of XLA in males and identifying carrier females requires testing for Btk protein expression on B cells by flow cytometry and genetic testing for a BTK mutation. Patients can be screened for the presence of Btk protein by flow cytometry (BTK/89011 Bruton's Tyrosine Kinase [Btk], Protein Expression, Flow Cytometry, Blood); however, normal results by flow cytometry do not rule out the presence of a BTK mutation with normal protein expression but aberrant protein function. The diagnosis is confirmed only in those individuals with appropriate clinical history who have a mutation identified in BTK by gene sequencing or who have male family members with hypogammaglobulinemia with absent or low B cells.

An interpretive report will be provided.

A patient-specific interpretive report is provided.

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 Bruton’s tyrosine kinase gene (BTK) gene or large rearrangement-type mutations (which could cause a false-negative result).

If the full gene sequencing does not match the clinical impression, flow cytometry should be performed to assess expression of Btk protein (BTK/89011 Bruton's Tyrosine Kinase [Btk], Protein Expression, Flow Cytometry, Blood). Large deletions or rearrangements will affect protein expression, and the absence of Btk protein on monocytes can be determined by flow cytometry.

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 Jan 29;72(2):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 healthy children. Pediatr Res 2002 Feb;51(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 Feb;203:216-234

4. Lindvall JM, Blomberg KE, Valiaho J, et al: Bruton’s tyrosine kinase: cell biology, sequence conservation, mutation spectrum, siRNA modifications, and expression profiling. Immunol Rev 2005 Feb;203:200-215

5. Valiaho J, Smith CI, Vihinen M: BTKbase: the mutation database for X-linked agammaglobulinemia. Hum Mutat 2006 Dec;27(12):1209-1217

6. Takada H, Kanegane H, Nomura A, et al: Female agammaglobulinemia due to the Bruton tyrosine kinase deficiency caused by extremely skewed X-chromosome inactivation. Blood 2004 Jan 1;103(1):185-187

Genomic DNA is first extracted from whole blood, followed by Bruton’s tyrosine kinase gene (BTK) amplification by PCR. 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 are analyzed for variations in the exons and intron/exon boundaries of all 19 exons using specialized mutation detection software (Phredphrap/Consed/Polyphred) and visual inspection. (Unpublished Mayo method)

Monday

81479 -Unlisted molecular pathology procedure