Test Code BTKSG Bruton Tyrosine Kinase, BTK Full Gene Analysis, Varies

Ordering Guidance

Targeted testing for familial variants (also called site-specific or known variants testing) is available for variants identified in the BTK gene. See FMTT / Familial Variant, Targeted Testing, Varies. To obtain more information about testing option, call 800-533-1710.

Additional Testing Requirements

To confirm a diagnosis of X-linked agammaglobulinemia in male patients, the preferred approach is to order this test concurrently with BTK / Bruton Tyrosine Kinase, Protein Expression, Flow Cytometry, Blood.

Shipping Instructions

Specimen preferred to arrive within 96 hours of collection.

Necessary Information

Bruton Tyrosine Kinase (BTK) Gene Sequencing Patient Information form (T620) is highly recommended. Testing may proceed without the patient information. However, it aids in providing a more thorough interpretation. Ordering providers are strongly encouraged to complete the form and send it with the specimen.

Specimen Required

Patient Preparation: A previous bone marrow transplant from an allogenic donor will interfere with testing. Call 800-533-1710 for instructions for testing patients who have received a bone marrow transplant.

Submit only 1 of the following specimens:

Specimen Type: Whole blood

Container/Tube:

Preferred: Lavender top (EDTA) or yellow top (ACD)

Acceptable: Any anticoagulant

Specimen Volume: 3 mL

Collection Instructions:

1. Invert several times to mix blood.

2. Send whole blood specimen in original tube. Do not aliquot.

Specimen Stability Information: Ambient (preferred) 4 days/Refrigerated

Specimen Type: Skin biopsy

Supplies: Fibroblast Biopsy Transport Media (T115)

Container/Tube: Sterile container with any standard cell culture media (eg, minimal essential media, RPMI 1640). The solution should be supplemented with 1% penicillin and streptomycin.

Specimen Volume: 4-mm punch

Specimen Stability Information: Refrigerated (preferred)/Ambient

Additional Information: A separate culture charge will be assessed under CULFB /Fibroblast Culture for Biochemical or Molecular Testing. An additional 3 to 4 weeks is required to culture fibroblasts before genetic testing can occur.

Specimen Type: Cultured fibroblasts

Container/Tube: T-25 flask

Specimen Volume: 2 Flasks

Collection Instructions: Submit confluent cultured fibroblast cells from a skin biopsy from another laboratory. Cultured cells from a prenatal specimen will not be accepted.

Specimen Stability Information: Ambient (preferred)/Refrigerated (<24 hours)

Forms

1. New York Clients-Informed consent is required. Document on the request form or electronic order that a copy is on file. The following documents are available:

-Informed Consent for Genetic Testing (T576)

-Informed Consent for Genetic Testing (Spanish) (T826)

2. Bruton Tyrosine Kinase (BTK) Gene Sequencing Patient Information form (T620)

Secondary ID

619760

Useful For

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

Evaluating for the presence of BTK variants in family members of affected individuals, including those who do not demonstrate carrier phenotype by BTK flow cytometry

Disease States

Common variable immunodeficiency

Reflex Tests

Test ID	Reporting Name	Available Separately	Always Performed
CULFB	Fibroblast Culture for Genetic Test	Yes	No

Testing Algorithm

For skin biopsy or cultured fibroblast specimens, fibroblast culture will be performed at an additional charge. If viable cells are not obtained, the client will be notified.

Special Instructions

Method Name

Sequence Capture and Targeted Next-Generation Sequencing (NGS) followed by Polymerase Chain Reaction (PCR) and Sanger Sequencing

Reporting Name

BTK Gene, Full Gene Analysis

Specimen Type

Varies

Specimen Minimum Volume

Blood: 1 mL; Skin biopsy or cultured fibroblasts: See Specimen Required

Specimen Stability Information

Specimen Type	Temperature	Time	Special Container
Varies	Varies

Reject Due To

All specimens will be evaluated at Mayo Clinic Laboratories for test suitability.

Clinical Information

X-linked agammaglobulinemia (XLA) is a humoral primary immunodeficiency affecting male patients in approximately 1 in 200,000 live births. XLA is caused by variants in the Bruton tyrosine kinase gene (BTK), 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.(1,2)

Approximately 85% of male patients with defects in early B-cell development have XLA. 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.(2) 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. XLA typically manifests in male infants.(2) However, other patients present with milder phenotypes, resulting in diagnosis later in childhood or in adulthood. Delayed diagnoses can be partly explained by the variable severity of XLA, even within families in which the same variant is present. X-inactivation of this gene is not typical, and XLA in female patients has rarely been reported.(3) Therefore, female patients with clinical features that are identical to XLA should be first evaluated for autosomal recessive agammaglobulinemia and for XLA if their biological father is affected with the disease.

A diagnosis of XLA should be suspected in male patients with early-onset bacterial infections, marked reduction in all classes of serum immunoglobulins, and absent B cells (CD19+ cells). The decrease in numbers of peripheral B cells is a key feature, although this can also be seen in a small subset of patients with common variable immunodeficiency. Conversely, some BTK variants can preserve small numbers of circulating B cells and, therefore, all 3 of the criteria mentioned above need to be evaluated.(2)

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

Reference Values

An interpretive report will be provided.

Interpretation

All detected variants are evaluated according to American College of Medical Genetics and Genomics recommendations.(4) Variants are classified based on known, predicted, or possible pathogenicity and reported with interpretive comments detailing their potential or known significance.

Method Description

Next-generation sequencing (NGS) and/or Sanger sequencing are performed to test for the presence of variants in coding regions and intron/exon boundaries of the BTK gene, as well as some other regions that have known disease-causing variants. The human genome reference GRCh37/hg19 build was used for sequence read alignment. At least 99% of the bases are covered at a read depth over 30X. Sensitivity is estimated at above 99% for single nucleotide variants, above 94% for deletions/insertions (delins) less than 40 base pairs (bp), and above 95% for deletions up to 75 bp and insertions up to 47 bp. NGS and/or a polymerase chain reaction-based quantitative method is performed to test for the presence of deletions and duplications in the BTK gene.

There may be regions of BTK that cannot be effectively evaluated by sequencing or deletion and duplication analysis as a result of technical limitations of the assay, including regions of homology, high guanine-cytosine (GC) content, and repetitive sequences.(Unpublished Mayo method)

Confirmation of select reportable variants may be performed by alternate methodologies based on internal laboratory criteria.

Reference transcript numbers may be updated due to transcript re-versioning. Always refer to the final patient report for gene transcript information referenced at the time of testing.

Gene symbol	Reference transcript	Additional evaluations	Technical limitations
BTK	NM_000061.2	c.-193A>G c.142-205A>G c.240+108T>G c.240+109C>A c.895-11C>A c.1102+2_1102+12del c.1177+26_1177+27insGGTAGAAAAAA c.1567-23A>C c.1567-23A>G c.1567-12_1567-9del	-

Day(s) Performed