Genotype -Phenotype Correlation of PKLR Variants with Pyruvate Kinase, 2,3-Diphosphglycerate and ATP Activities in Red Blood Cells of Patients with Sickle Cell Disease

Updated on 22 December 2020
sickle cell disease
sickle cell anemia
red blood cell disorder



  • Some people with the same disorder on a genetic level have more complications than others. Researchers want to look for a link between the PKLR gene and sickle cell disease (SCD) symptoms. The PKLR gene helps create a protein, called pyruvate kinase that is essential in normal functioning of the red blood cell. Differences in the PKLR gene, called genetic variants, may cause some changes in the pyruvate kinase protein and other proteins, that can affect functioning of the red blood cell adding to the effect of SCD. Researchers can study these differences by looking at DNA (the material that determines inherited characteristics).


  • To study how the PKLR gene affects sickle cell disease.


  • Adults ages 18-80 of African descent. They may have sickle cell disease or not. They must not have had a transfusion recently or have a known deficiency of pyruvate kinase. They cannot be pregnant.


  • Participants will be screened with questions.
  • Participants will have blood drawn by needle in an arm vein. The blood will be genetically tested. Not much is known about how genes affect SCD, so the test results will not be shared with participants or their doctors.

Please visit our Patient Recruitment Page for more information.



Polymerization of deoxy-sickle-hemoglobin (deoxy-HbS), the root cause of sickle cell disease (SCD) is influenced by a few factors, a key factor is 2,3-diphosphoglycerate (2,3-DPG) concentration in the red blood cells. 2,3-DPG is an allosteric effector on hemoglobin oxygen binding with a greater binding affinity to deoxygenated hemoglobin than to oxygenated hemoglobin, thus favoring polymerization of deoxy-HbS. In addition, increased 2,3-DPG concentration decreases intracellular pH in red blood cells which further promotes HbS polymerization.

2,3-DPG is an intermediate substrate in the glycolytic pathway, the only source of ATP production in red blood cells. Pyruvate kinase (PK) is a key enzyme in the final step of glycolysis; PK converts phosphoenolpyruvate (PEP) to pyruvate, creating 50% of the total red cell adenosine triphosphate (ATP) that is essential for maintaining integrity of the red cell membrane. Indeed, PK deficiency (PKD) caused by mutations in the PKLR gene that encodes red cell PK, leads to chronic hemolytic anemia. Reduced PK activity leads to accumulation of the upstream enzyme substrates, including 2,3-DPG. While increased 2,3-DPG concentration and reduction of hemoglobin oxygen affinity is beneficial in anemia caused by PKD, increased 2,3-DPG levels combined with decreased intracellular red cell pH can be detrimental in the presence of HbS, as it favors deoxy-HbS polymerisation, and thereby intravascular sickling. Indeed, the combination of PK deficiency and sickle cell trait causing an acute sickling syndrome has been previously reported in two cases.

PKLR mutations, however, are rare but intraerythrocytic PK enzyme levels form a spectrum which suggest that PKLR is likely to be a quantitative trait gene. A genetic diversity in PKLR with a range of SNPs, including several loss-of-function variants have been described in malaria-endemic populations, some of which have been associated with a significant reduction in attacks with Plasmodium falciparum malaria. These observations suggest that similar to HbS, malaria has led to positive selection of PKLR variants in the same geographic regions.

This study seeks to determine the PKLR genetic diversity in our sickle cell cohort, and whether PKLR variants modify PK levels, and activities of 2,3-DPG and ATP, key players in the sickle pathology. If so, PKLR could be another genetic determinant of SCD severity and phenotype; and increasing PK-R activity, which leads to a decrease in intracellular 2,3-DPG concentration, presents an attractive therapeutic target for SCD.

Several approaches have been considered for targeting the polymerization of deoxy-HbS, the root cause of SCD. In addition to agents inducing fetal hemoglobin, other agents that target HbS polymerization through increasing affinity of hemoglobin for oxygen (eg. GBT440), are in clinical trials (NCT03036813; NCT02850406). The results of this study could form the basis for a clinical trial of AG348, an allosteric activator of PK that is already in clinical Phase 2/3 studies for PK deficiency (NCT02476916), for treating acute sickle cell pain.


Please visit our Patient Recruitment Page for more information.

Condition Red Blood Cell Disorders, Sickle Cell Disease, Sickle Cell Disease Sickle Cell PKLR Variants Adenosine Triphosphate Activities
Clinical Study IdentifierTX218212
Last Modified on22 December 2020


Yes No Not Sure

Inclusion Criteria

Between 18 and 80 years of age
African or of African descent
Willingness and capacity to provide written informed consent

Exclusion Criteria

History of blood transfusion within the last 8 weeks
Known to have pyruvate kinase deficiency and be on AG348
All volunteers will undergo the consent process under this protocol to allow for eligibility assessment. Once they have been consented to participate, they will undergo procedures per section 6.0
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