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

National Heart, Lung, And Blood Institute

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Abstract

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 (Eaton and Bunn 2017). 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 (Rose and Warms 1966). 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 (Grace et al, 2015). 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 (Charache et al, 1970). Indeed, the combination of PK deficiency and sickle cell trait causing an acute sickling syndrome has been previously reported in two cases (Alli et al, 2008; Cohen-Solal et al, 1998). 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 (Berghout et al, 2012; van Bruggen et al, 2015). 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. Enrolment for this protocol started on 11 Oct 2018, and has been actively accruing subjects - healthy African-Americans, Individuals who are carriers for SCD (AS) and individuals with SCD. Our accrual ceiling is 780 (total) with the goal of completing 250 in each cohort of AA, AS and SCD. An aggregate total of 654 has been recruited, 251 AA, 177 AS and 226 SS. DNA extraction, genotyping and assays for pyruvate kinase, 2,3-diphosphoglycerate and ATP are being performed as samples accrue.