Pyruvate Kinase Deficiency with Hemolytic Anemia via the PKLR Gene

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
1652 PKLR$840.00 81479 Add to Order
Targeted Testing

For ordering targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 18 days.

Clinical Sensitivity

In patients with biochemical evidence indicating pyruvate kinase deficiency, 58 of 60 and 53 of 58 had mutations within the PKLR gene (Baronciani and Beutler 1995; Lenzner et al. 1997). Analytical sensitivity for identifying PKLR mutations by this sequencing method is >95% as large deletions have been reported only in a few cases.

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 PKLR$690.00 81479 Add to Order
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Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Features

Pyruvate kinase deficiency (PKD) is a relatively common enzymatic defect in red blood cells affecting an estimated one in 20,000 people and is the leading cause of hereditary nonspherocytic hemolytic anemia (Beutler and Gelbart 2000). Patients affected with this chronic hemolytic anemia exhibit pale skin, jaundice, fatigue, dyspnea, and tachycardia. Splenomegaly, excess iron in the blood, and gall stones are also common symptoms associated with PKD. Disease severity ranges from life threatening in infancy requiring regular blood transfusions to asymptomatic. Symptoms may be exacerbated by underlying infections. Other disorders have been associated with inherited hemolytic anemia including hereditary spherocytosis (via the ANK1, SPTB, SPTA1, EPB42, and SLC4A1 genes) and glucose-6-phosphate deficiency (via the G6PD gene) (Frank 2005; An and Mohandas 2008). PKD may be masked by reticulocytosis. Symptoms are similar to other forms of congenital hemolytic anemia. Genetic testing is helpful for differential diagnosis of distinct congenital hemolytic anemias and for distinguishing between inherited and acquired forms of the disease (Vercellati et al. 2013). Treatment for PKD includes blood transfusions, folic acid supplementation and in severe cases bone marrow transplantation or splenectomy (Zanella et al. 2005).


PKD is inherited in an autosomal recessive manner through mutation in the PKLR gene. The PKLR gene uses alternative promoters to encode the shorter liver specific and longer red blood cell specific isoforms. Mutations identified are found throughout the coding region with missense (65%), splicing (11%), and nonsense (5%) being most prevalent (Zanella et al. 2005). Frameshift and small indel mutations make up 12% of causative variants. Gross deletions have been reported in a minority of cases with the Gypsy deletion, loss of exon 11, and PK ‘Viet’, loss of exons 4-10, being most common (Baronciani and Beutler 1995; Fermo et al 2005). Two substitution mutations have been found within the promoter region leading to disruption of GATA1 transcription factor binding (c.-72C>G) and alteration of the PKLR regulatory element (c.-83G>C ) (Manco et al. 2000; van Wijk et al. 2003). Founder missense mutations play an important role with the c.1529G>A resulting in p.Arg510Gln variant being found in ~40% of North American and Central European patients (Baronciani and Beutler 1995; Lenzer et al. 1997). The c.1456C>T mutation resulting in p.Arg486Trp is found in ~30% of Southern Europeans. The PKRL gene encodes pyruvate kinase which catalyzes the transphosphorylation of phosphoenolpyruvate to ADP yielding ATP and pyruvate. This metabolic reaction is the last step in glycolysis and is essential for providing ATP energy to red blood cells, which rely heavily on glycolysis for energy due to their lack of mitochondria (Zanella et al. 2005).

Testing Strategy

This test involves bidirectional Sanger sequencing using genomic DNA of all coding exons of the PKLR gene plus ~20 bp of flanking non-coding DNA on each side and 100 bp upstream of the initiating codon. We will also sequence any single exon (Test #100) or pair of exons (Test #200) in family members with known mutations or to confirm research results.

Indications for Test

Candidates for this test are patients showing features consistent with PKD (anemia, increased LDH, decreased haptoglobin and jaundice. Ideal candidates have biochemical results indicating impaired pyruvate kinase enzymatic activity and a family history for the disorder (Aster et al. 2013). Unlike congenital spherocytic hemolytic anemias, red blood cells are non-spheroid and osmotic fragility is normal.


Official Gene Symbol OMIM ID
PKLR 609712
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT


Name Inheritance OMIM ID
Pyruvate Kinase Deficiency 266200


Genetic Counselors
  • An X, Mohandas N. 2008. Disorders of red cell membrane. Br. J. Haematol. 141: 367–375. PubMed ID: 18341630
  • Aster, JC, Pozdnyakova, O, Kutok, JL. Hematopathology. Philadelphia: Elsevier Saunders, 2013.
  • Baronciani L, Beutler E. 1995. Molecular study of pyruvate kinase deficient patients with hereditary nonspherocytic hemolytic anemia. J. Clin. Invest. 95: 1702–1709. PubMed ID: 7706479
  • Beutler E, Gelbart T. 2000. Estimating the prevalence of pyruvate kinase deficiency from the gene frequency in the general white population. Blood 95: 3585–3588. PubMed ID: 10828047
  • Fermo E, Bianchi P, Chiarelli LR, Cotton F, Vercellati C, Writzl K, Baker K, Hann I, Rodwell R, Valentini G, Zanella A. 2005. Red cell pyruvate kinase deficiency: 17 new mutations of the PK-LR gene. Br. J. Haematol. 129: 839–846. PubMed ID: 15953013
  • Frank JE. 2005. Diagnosis and management of G6PD deficiency. Am Fam Physician 72: 1277–1282. PubMed ID: 16225031
  • Lenzner C, Nürnberg P, Jacobasch G, Gerth C, Thiele BJ. 1997. Molecular analysis of 29 pyruvate kinase-deficient patients from central Europe with hereditary hemolytic anemia. Blood 89: 1793–1799. PubMed ID: 9057665
  • Manco L, Ribeiro ML, Máximo V, Almeida H, Costa A, Freitas O, Barbot J, Abade A, Tamagnini G. 2000. A new PKLR gene mutation in the R-type promoter region affects the gene transcription causing pyruvate kinase deficiency. Br. J. Haematol. 110: 993–997. PubMed ID: 11054094
  • Vercellati C, Marcello AP, Fermo E, Barcellini W, Zanella A, Bianchi P. 2013. A case of hereditary spherocytosis misdiagnosed as pyruvate kinase deficient hemolytic anemia. Clin. Lab. 59: 421–424. PubMed ID: 23724634
  • Wijk R van, Solinge WW van, Nerlov C, Beutler E, Gelbart T, Rijksen G, Nielsen FC. 2003. Disruption of a novel regulatory element in the erythroid-specific promoter of the human PKLR gene causes severe pyruvate kinase deficiency. Blood 101: 1596–1602. PubMed ID: 12393511
  • Zanella A, Fermo E, Bianchi P, Valentini G. 2005. Red cell pyruvate kinase deficiency: molecular and clinical aspects. Br. J. Haematol. 130: 11–25. PubMed ID: 15982340
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Bi-Directional Sanger Sequencing

Test Procedure

Nomenclature for sequence variants was from the Human Genome Variation Society (  As required, DNA is extracted from the patient specimen.  PCR is used to amplify the indicated exons plus additional flanking non-coding sequence.  After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions.  In nearly all cases, the full coding region of each exon as well as 20 bases of non-coding DNA flanking the exon are sequenced.

Analytical Validity

As of March 2016, we compared 17.37 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 12 years of our lab operation we have Sanger sequenced roughly 8,800 PCR amplicons. Only one error has been identified, and this was due to sequence analysis error.

Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).

Analytical Limitations

In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.

Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.

In most cases, only the indicated exons and roughly 20 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.

In nearly all cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants, due for example to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR and cycle sequencing.

Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.

Deletion/Duplication Testing via Array Comparative Genomic Hybridization

Test Procedure

Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are extracted and analyzed.

Analytical Validity

PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. PreventionGenetics has established and verified this test's accuracy and precision.

Analytical Limitations

Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.

This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.

aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.

Breakpoints, if occurring outside the targeted gene, may be hard to define.

The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.

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Ordering Options

myPrevent - Online Ordering
  • The test can be added to your online orders in the Summary and Pricing section.
  • Once the test has been added log in to myPrevent to fill out an online requisition form.
  • A completed requisition form must accompany all specimens.
  • Billing information along with specimen and shipping instructions are within the requisition form.
  • All testing must be ordered by a qualified healthcare provider.


(Delivery accepted Monday - Saturday)

  • Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
  • For small babies, we require a minimum of 1 ml of blood.
  • Only one blood tube is required for multiple tests.
  • Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
  • During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
  • In cold weather, include an unfrozen ice pack in the shipping container as insulation.
  • At room temperature, blood specimen is stable for up to 48 hours.
  • If refrigerated, blood specimen is stable for up to one week.
  • Label the tube with the patient name, date of birth and/or ID number.


(Delivery accepted Monday - Saturday)

  • Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
  • For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
  • DNA may be shipped at room temperature.
  • Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
  • We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.


(Delivery preferred Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Culture and send at least two T25 flasks of confluent cells.
  • Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
  • Send specimens in insulated, shatterproof container overnight.
  • Cell cultures may be shipped at room temperature or refrigerated.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We strongly recommend maintaining a local back-up culture. We do not culture cells.
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