Genotype -Phenotype Correlation of PKLR Variants With Pyruvate Kinase, 2,3-Diphosphglycerate and Adenosine Triphosphate Activities in Red Blood Cells of People With Sickle Cell Disease

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.

HbAS genotype, of African American descent;Between 18 and 80 years of age

African American descent;Between 18 and 80 years of age

HbSS, HbSC, HbSbeta-thal has sickle cell disease and is of African American descent;Between 18 and 80 years of age

Time frame: Upon enrollment of each subject

To have genotyped the 4 PKLR intron-2 variants in SCD patients from the NHLBI cohort using genomic DNA and compare them to a cohort of healthy ethnic-matched non-SCD controls and a cohort of sickle cell trait carriers, with those reported in 1000 genome project (http://www.1000genomes.org).

Time frame: Interim analysis performed for each group N=125

Have a correlated profile of the PK-R RNA sequence with the 4 PKLR intronic genetic variants.

Time frame: Interim analysis performed for each group N=125

Assess correlation between the quantitative assays and genotype

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