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

- INCLUSUION 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.