Test Id : PKLRZ

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.

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 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

• Informed Consent for Genetic Testing
• PKLR Gene Sequencing Patient Information
• Informed Consent for Genetic Testing (Spanish)

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)

1 mL

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

This test utilizes next-generation sequencing to detect single nucleotide and copy number variants in PKLR. See Method Description for additional details.

Identification of a disease-causing variant may assist with diagnosis, prognosis, clinical management, recurrence risk assessment, familial screening, and genetic counseling for pyruvate kinase deficiency.

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.

An interpretive report will be provided.

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.

Clinical Correlations:

Test results should be interpreted in the context of clinical findings, family history, and other laboratory data. Misinterpretation of results may occur if the information provided is inaccurate or incomplete.

If testing was performed because of a clinically significant family history, it is often useful to first test an affected family member. Detection of a reportable variant in an affected family member would allow for more informative testing of at-risk individuals.

To discuss the availability of additional testing options or for assistance in the interpretation of these results, contact the Mayo Clinic Laboratories genetic counselors at 800-533-1710.

If the patient has had an allogeneic hematopoietic stem cell transplant or a recent heterologous blood transfusion, results may be inaccurate due to the presence of donor DNA. Call Mayo Clinic Laboratories for instructions for testing patients who have received a bone marrow transplant.

For individuals who have received blood transfusions, the genotype usually reverts to that of the of the recipient within 6 weeks. For individuals who have received allogeneic blood or marrow transplantation, a pretransplant DNA specimen is recommended for testing. For patients who have been transfused within the preceding 6 weeks, the enzyme assay (PK1 / Pyruvate Kinase Enzyme Activity, Blood) will also be affected, so it is not an appropriate alternative test.

Patients who have received an allogeneic blood or marrow transplant would be expected to convert to the PKLR status of the donor. However, if the patient's transplant was partially successful or if there is a relapse of an underlying hematologic malignancy, a mixture of donor and recipient genotypes may be seen on genetic analysis. The enzyme assay can be performed after transplantation; order PK1 / Pyruvate Kinase Enzyme Activity, Blood.

Technical Limitations:

Next-generation sequencing may not detect all types of genomic variants. In rare cases, false-negative or false-positive results may occur. The depth of coverage may be variable for some target regions; assay performance below the minimum acceptable criteria or for failed regions will be noted. Given these limitations, negative results do not rule out the diagnosis of a genetic disorder. If a specific clinical disorder is suspected, evaluation by alternative methods can be considered.

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

This test is validated to detect 95% of deletions up to 75 base pairs (bp) and insertions up to 47 bp. Deletions-insertions (delins) of 40 or more bp, including mobile element insertions, may be less reliably detected than smaller delins.

Deletion/Duplication Analysis:

This analysis targets single and multiexon deletions/duplications; however, in some instances, single exon resolution cannot be achieved due to isolated reduction in sequence coverage or inherent genomic complexity. Balanced structural rearrangements (such as translocations and inversions) may not be detected.

This test is not designed to detect low levels of mosaicism or to differentiate between somatic and germline variants. If there is a possibility that any detected variant is somatic, additional testing may be necessary to clarify the significance of results.

For detailed information regarding gene-specific performance and technical limitations, see Method Description or contact a laboratory genetic counselor.

Reclassification of Variants:

Currently, it is not standard practice for the laboratory to systematically review previously classified variants on a regular basis. The laboratory encourages healthcare professionals to contact the laboratory at any time to learn how the classification of a particular variant may have changed over time. Due to broadening genetic knowledge, it is possible that the laboratory may discover new information of relevance to the patient. Should that occur, the laboratory may issue an amended report.

Variant Evaluation:

Evaluation and categorization of variants are performed using published American College of Medical Genetics and Genomics and the Association for Molecular Pathology recommendations as a guideline.(3) Other gene-specific guidelines may also be considered. Variants are classified based on known, predicted, or possible pathogenicity and reported with interpretive comments detailing their potential or known significance. Variants classified as benign or likely benign or intronic variants without known or suspected pathogenicity are not reported.

Multiple in silico evaluation tools may be used to assist in the interpretation of these results. The accuracy of predictions made by in silico evaluation tools is highly dependent upon the data available for a given gene, and periodic updates to these tools may cause predictions to change over time. Results from in silico evaluation tools should be interpreted with caution and professional clinical judgment.

Rarely, incidental or secondary findings may implicate another predisposition or presence of active disease. These findings will be carefully reviewed to determine whether they will be reported.

1. van Wijk R, Huizinga E, van Wesel AC, et al. Fifteen novel mutations in PKLR associated with pyruvate (PK) deficiency: structural implications of amino acid substitutions in PK. Hum Mutat. 2009;30(3):446-453

2. Zanella A, Fermo E, Bianchi P, et al. Pyruvate kinase deficiency: the genotype-phenotype association. Blood Rev. 2007;21(4):217-231

3. Richards S, Aziz N, Bale S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17(5):405-424

4. OMIM: 609712 Pyruvate Kinase, Liver and Red Blood Cell; PKLR. Johns Hopkins University; 2005. Updated November 2022. Accessed January 6, 2023. Available at www.omim.org/entry/609712

5. Baronciani L, Beutler E. Molecular study of pyruvate deficient patients with hereditary nonspherocytic hemolytic anemia. J Clin Invest. 1995;95(4):1702-1709

6. Bianchi P, Zanella A. Hematologically important mutations: red cell pyruvate kinase (Third update). Blood Cells Mol Dis. 2000;26(3):47-53

7. Costa C, Albuisson J, Le TH, et al. Severe hemolytic anemia in a Vietnamese family, associated with novel mutations in the gene encoding for pyruvate kinase. Haematologica. 2005;90(1):25-30

8. So CC, Tang M, Li CH, et al. First reported case of prenatal diagnosis for pyruvate kinase deficiency in a Chinese family. Hematology. 2011;16(6):377-379

9. van Wijk R, van Solinge WW, Nerlov C, et al. Disruption of a novel regulatory element in the erythroid-specific promoter of the human PKLR gene causes severe pyruvate kinase deficiency. Blood. 2003;101(4):1596-1062

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

Varies

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This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. It has not been cleared or approved by the US Food and Drug Administration.

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