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
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
Background:

 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).

 Objective:

 To study how the PKLR gene affects sickle cell disease.

 Eligibility:

 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.

 Design:

 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.

 ...
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 Protocol.