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 ATP Activities in Red Blood Cells of Patients 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.

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Study Overview

• Status: Recruiting
• Conditions: Sickle Cell; PKLR Variants; Adenosine Triphosphate Activities

Detailed Description

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.

• Study Type: Observational
• Enrollment (Estimated): 800

Contacts and Locations

• Study Contact: Name: Ingrid C Frey; Phone Number: (301) 221-3820; Email: ingrid.frey@nih.gov

Study Locations

• United States
  • Maryland
    • Bethesda, Maryland, United States, 20892
      • Recruiting
      • National Institutes of Health Clinical Center
      • Contact: For more information at the NIH Clinical Center contact Office of Patient Recruitment (OPR); Phone Number: TTY8664111010 800-411-1222; Email: prpl@cc.nih.gov

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 18 years to 80 years (Adult, Older Adult)
• Accepts Healthy Volunteers: Yes

• Sampling Method: Non-Probability Sample

Study Population

The study will be listed on the clinicaltrials.gov, Clinical Center research studies, and the National Heart, Lung and Blood Institute patient recruitment websites. Patients who are followed on other NHLBI sickle cell protocols may be asked to participate in this study, particularly subjects enrolled in the Natural History of Sickle Cell Disease (NCT00081523; 04-H-0161).

Description

• INCLUSUION CRITERIA:
• Between 18 and 80 years of age
• African or of African descent

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.

Study Plan

How is the study designed?

Design Details

• Observational Models: Case-Control
• Time Perspectives: Cross-Sectional

Number of groups / cohorts

3

Cohorts and Interventions

Group / Cohort
HbAS; HbAS genotype, of African American descent;Between 18 and 80 years of age
Healthy control; African American descent;Between 18 and 80 years of age
SCD; HbSS, HbSC, HbSbeta-thal has sickle cell disease and is of African American descent;Between 18 and 80 years of age

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Genotype the 4 PKLR intron-2 variants	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).	Upon enrollment of each subject
Analysis of PK-R transcriptome in red blood cells	Have a correlated profile of the PK-R RNA sequence with the 4 PKLR intronic genetic variants.	Interim analysis performed for each group N=125
Correlation of 2,3-DPG, ATP and pyruvate kinase activities with PKLR intron-2 variants	Assess correlation between the quantitative assays and genotype	Interim analysis performed for each group N=125

Collaborators and Investigators

• Sponsor: National Heart, Lung, and Blood Institute (NHLBI)
• Investigators: Principal Investigator: Swee Lay Thein, M.D., National Heart, Lung, and Blood Institute (NHLBI)

Publications and helpful links

Helpful Links

• NIH Clinical Center Detailed Web Page

Study record dates

Study Major Dates

• Study Start (Actual): October 11, 2018
• Primary Completion (Estimated): July 25, 2024
• Study Completion (Estimated): May 1, 2025

Study Registration Dates

• First Submitted: September 25, 2018
• First Submitted That Met QC Criteria: September 25, 2018
• First Posted (Actual): September 26, 2018

Study Record Updates

• Last Update Posted (Actual): March 21, 2024
• Last Update Submitted That Met QC Criteria: March 20, 2024
• Last Verified: February 22, 2024

More Information

Terms related to this study

• Keywords: Genetics; Natural History; ATP; Trait; GDP
• Additional Relevant MeSH Terms: Hematologic Diseases; Genetic Diseases, Inborn; Anemia; Anemia, Hemolytic, Congenital; Anemia, Hemolytic; Hemoglobinopathies; Anemia, Sickle Cell
• Other Study ID Numbers: 180146; 18-H-0146

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No