Polymerase Chain Reaction/Sequencing
Molecular confirmation of suspected PK deficiency in individuals with abnormal PK enzyme activity and/or clinical findings. Assess carrier status for PK deficiency.
Massively Parallel Sequencing
Determine etiology of unexplained hemolytic anemia or family history of unexplained hemolytic anemia. Determine etiology of unexplained hyperbilirubinemia in neonates.
Red cell pyruvate kinase (PK) deficiency, although relatively rare, is the most common glycolytic defect resulting in congenital nonspherocytic hemolytic anemia (CNSHA). The PKLR gene produces PK in the liver and red blood cells (RBCs) that converts phosphoenolpyruvate to pyruvate, creating 50% of the red cell adenosine triphosphate (ATP). Pathogenic variants in PKLR cause reduced PK function, leading to the accumulation of intermediate glycolysis by-products and a shortage of ATP in RBCs. This results in shortened RBC lifespan and damaged cells are removed from circulation by the spleen. Clinical features of PK deficiency are highly variable, ranging from well-compensated anemia to severe disease with lifelong transfusion dependency. Other clinical manifestations may include jaundice, gallstones, iron overload, and potential for other complications.
Typical testing strategy includes PK activity level followed by molecular testing to confirm diagnosis in individuals with reduced PK activity and/or clinical findings. Molecular testing is the most reliable method of identifying heterozygous PKLR variant carriers. Carriers often have intermediate levels of PK activity, but are not at risk for clinical symptoms.
Prenatal growth restriction
- Most newborns are treated with phototherapy; many require exchange transfusion
Chronic hemolytic anemia of varying severity
- Infants and young children may be transfusion-dependent prior to splenectomy
- Anemia may stabilize in adulthood; however, exacerbations can result with infections, pregnancy, or stress
- 2,3 diphosphoglycerate is elevated and shifts oxygen dissociation curve to favor unloading of oxygen in tissues, thus, anemia may be better tolerated than in other conditions
- Increase may not be proportional to severity of anemia
Reduced red cell PK activity
- Contamination with normal donor RBCs in transfused patients or compensatory persistence of the M2 fetal isoform may occasionally result in normal PK activity
- Iron overload
- Less common: aplastic crises, osteopenia/bone fragility, extramedullary hematopoiesis, postsplenectomy sepsis, pulmonary hypertension, or leg ulcers
- Splenectomy may moderately improve anemia and reduce transfusion burden
PKLR sequencing: polymerase chain reaction (PCR) followed by bidirectional sequencing of all coding regions and intron-exon boundaries, 5’ untranslated region, and deep intronic variants c.1269+43T>C and c.1269+44C>T (also known as IVS9+43T>C and IVS9+44C>T, respectively)
Over 250 disease-associated PKLR variants have been described:
- c.1529G>A: common variant in U.S. and Europe
- c.1456C>T: common variant in Southern Europe, homozygosity associated with mild phenotype
- c.1468C>T: common variant in Asia
- c.1436G>A: Pennsylvania Amish founder variant
- 1,149 bp deletion: Romani founder variant known as “PK Gypsy” (not detectable by sequencing alone)
PK enzyme activity is not correlated with genotype.
Individuals with two causative missense variants have lower likelihood of splenectomy, fewer lifetime transfusions, and lower rate of iron overload versus individuals with nonmissense variants (ie, frameshift, nonsense, indels, large deletions, or splicing variants).
Two pathogenic PKLR variants on opposite chromosomes
- Consistent with a diagnosis of PK deficiency
One pathogenic PKLR variant identified
- At least a carrier of PK deficiency, may be affected if a second unidentified variant is present on opposite chromosome
No pathogenic variants identified
- Significantly reduces the likelihood of PK deficiency or carrier status
PKLR sequencing may identify variants of unknown clinical significance
Limitations of Sanger Sequencing
- Large deletions/duplications, including the Romani founder variant
- Repeat element insertions
- Deep intronic variants other than those targeted
- Regulatory region variants outside of the 5’UTR
Diagnostic errors can occur due to rare sequence variation
Beutler E, Gelbart T. Estimating the prevalence of pyruvate kinase deficiency from the gene frequency in the general white population. Blood. 2000;95(11):3585-3588.PubMed
Canu G, De Bonis M, Minucci A, et al. Red blood cell PK deficiency: An update of PK-LR gene mutation database. Blood Cells Mol Dis. 2016;57:100-109.
Grace RF, Bianchi P, van Beers EJ, et al. Clinical spectrum of pyruvate kinase deficiency: data from the Pyruvate Kinase Deficiency Natural History Study. Blood. 2018;131(20):2183-2192.