Pyruvate Kinase Deficiency with Hemolytic Anemia via the PKLR Gene

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).

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.

This test provides full coverage of all coding exons of the PKLR gene plus 10 bases of flanking noncoding DNA in all available transcripts along with other non-coding regions in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere. We define full coverage as >20X NGS reads or Sanger sequencing. PGnome panels typically provide slightly increased coverage over the PGxome equivalent. PGnome sequencing panels have the added benefit of additional analysis and reporting of deep intronic regions (where applicable).

Dependent on the sequencing backbone selected for this testing, discounted reflex testing to any other similar backbone-based test is available (i.e., PGxome panel to whole PGxome; PGnome panel to whole PGnome).