Test Code PKLRZ PKLR Full Gene Analysis, Varies

Ordering Guidance

Preliminary screening tests, such as complete blood cell count with peripheral smear, direct Coombs test, and pyruvate kinase enzyme activity assays (preferably as a part of EEEV1 / Red Blood Cell [RBC] Enzyme Evaluation, Blood) should be performed before ordering this test.

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

Necessary Information

1. PKLR Gene Sequencing Patient Information is required. Testing may proceed without the patient information; however, it aids in providing a more thorough interpretation. Ordering healthcare professionals are strongly encouraged to complete the form and send it with the specimen.

2. Include healthcare professional's name and phone number with specimen.

Specimen Required

Specimen Type: Whole blood

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

Container/Tube:

Preferred: Lavender top (EDTA)

Acceptable: Yellow top (ACD)

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

Forms

1. PKLR Gene Sequencing Patient Information (T766) is required.

2. 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)

Secondary ID

610058

Useful For

Aiding in the diagnosis of pyruvate kinase (PK) deficiency

Ascertaining a causative variant in the PKLR gene of patients with low or relatively low levels of erythrocytic PK enzymatic activity

Ascertaining carrier status of family members of individuals diagnosed with PK deficiency for genetic counseling purposes

Special Instructions

Method Name

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

Reporting Name

PKLR Full Gene Analysis

Specimen Type

Varies

Specimen Minimum Volume

1 mL

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

The glycolytic pathway is used by all tissues for energy production through the formation of adenosine triphosphate (ATP). It is particularly important in red blood cells, which are dependent upon this pathway for energy due to their lack of mitochondria. The PKLR gene encodes for pyruvate kinase (PK), the rate-limiting glycolytic enzyme that catalyzes the transphosphorylation from phosphoenolpyruvate to adenosine diphosphate, creating pyruvate and ATP.

Pyruvate kinase deficiency is a relatively common cause of hereditary nonspherocytic hemolytic anemia,(1) with an estimated prevalence of 1:20,000 among people of European descent. The severity of hemolysis varies from fully compensated forms to life-threatening neonatal anemia requiring transfusions.(2) Over 200 different variants have been reported in the PKLR gene. Most are single nucleotide substitutions, although rarer large deletions have also been identified. PK deficiency is inherited in an autosomal recessive manner, and genetic results should be correlated with enzyme levels performed as remote from transfusion when possible. PK deficiency can be difficult to interpret based on enzyme level alone and may be only mildly decreased or normal in those with the most severe symptoms or after splenectomy due to reticulocytosis.(2) Comparison to other erythrocyte enzyme levels is usually very helpful in this regard. Heterozygous carriers of PKLR variants have intermediate enzyme levels and are not expected to be symptomatic.

Reference Values

An interpretive report will be provided.

Interpretation

All detected variants are evaluated according to American College of Medical Genetics and Genomics recommendations.(3) 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 PKLR 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 20X. Sensitivity is estimated at above 99% for single nucleotide variants, above 94% for deletions-insertions (delins) less than 40 base pairs (bp), 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 PKLR gene.

There may be regions of this gene 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)

The reference transcript for PKLR gene is NM_000298.6. 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. Confirmation of select reportable variants may be performed by alternate methodologies based on internal laboratory criteria.

The following additional noncoding variants are being analyzed by this test:

±30 bp flanking exons, c.-98 to c.-1, c.1269+43T>C, NM_181871.3 Exon 1

Day(s) Performed