Background: We previously demonstrated association of PKLR intron 2 variants with hospitalization rate for acute pain in people with sickle cell disease (SCD) and utilized allele-specific expression analysis to show that these variants influence PKLR activity (Wang et al, 2022). Pyruvate kinase (gene PKLR, protein PKR) is a rate-limiting enzyme in glycolysis. Reduced PKR activity leads to an increase in intracellular 2,3-diphosphoglycerate (2,3-DPG), and a concomitant decrease of adenosine triphosphate (ATP), factors that promote sickle hemoglobin (HbS) polymerization and red blood cell (RBC) sickling. On this basis, we explored how the "high risk" PKLR intron 2 variants affect levels of PKR protein expression, 2,3-DPG, ATP, oxygen dissociation (p50) and sickling kinetics (t50). Methods: We studied a total of 416 subjects which included 125 HbSS, 89 HbAS, and 202 ethnic-matched healthy controls (HbAA) enrolled under protocol NCT03685721 approved by the NHLBI Institutional Review Board. DNA was genome scanned using Ilumina's Infinium "MEGA" chip (1.7m markers). The results were quality controlled followed by genotype imputation based on the 1000 Genomes Project phase 3 data using the Michigan Imputation Server. Whole blood levels of ATP and 2,3-DPG were measured using LC-MS/MS with LLOQ at 50.0 μg/mL and converted to intracellular concentrations by dividing by the hematocrit (as a fraction). PKR was detected by antibody-based capture and detection using an electrochemiluminescence immunoassay (Meso Scale Discovery, Rockville, MD, USA) and the signal normalized to a control sample. A Hemox Analyzer [TCS, Scientific Corp, New Hope, PA] was used to obtain p50 values (defined as the partial pressure of oxygen at which Hb is 50% saturated with oxygen). t50 is single parameter measure of sickling kinetics (Dunkelberger et al, 2018). Single variant analyses (under additive genetic model adjusted for age and sex) were conducted in each of the Hb sub-populations for associations between the 7 PKLR variants (intron 4 - rs2071053, and intron 2 -rs8177970, rs116244351, rs114455416, rs12741350, rs3020781, and rs8177964) and the 5 parameters (PKR, 2,3-DPG, ATP, p50 and t50). False-discovery rate (FDR) adjusted p-values were calculated to account for the multiple testing issue. Results: Table 1A shows levels of the 5 parameters (ATP/HCT, 2,3-DPG/HCT, PKR, p50 and t50) in the 3 Hb genotypic groups (HbAA, HbAS and HbSS). Because patients with SCD have variable anemia, ATP and 2,3-DPG were normalized against HCT. The observed trending higher ATP / HCT and significantly elevated 2,3-DPG /HCT in HbSS compared to HbAS and to HbAA are consistent with previous reports (Poillon et al, 1990). HbSS subjects also had higher PKR protein expression, as well as right-shifted p50 values, compared to HbAS and HbAA (Table 1A), which suggest HbSS RBCs have lower specific activity of PKR. A one-way ANOVA showed that the differences in these mean values were highly significant (Table 1A). All 7 PKLR variants were associated with ATP levels in the HbAS group, of which 4 (rs8177970, rs116244351, rs114455416 and rs8177964) in intron 2 remained statistically significant after multiple testing adjustment (Table 1B). We also identified association between 3 variants (intron 2 - rs12741350, rs3020781, intron 4 - rs071053) and 2,3-DPG levels in the HbSS, as well as t50 in the HbAS group. Conclusion: ATP is essential for maintaining RBC membrane integrity and shape; reduced ATP dehydrates RBCs and promotes sickling. In a previous report, a PKLR intron 2 'high-risk" haplotype was implicated in acute sickle pain by affecting PKLR expression (Wang et al, 2022). In this follow-up study, we show that the same intron 2 "high-risk" haplotype negatively affects ATP levels when accounting for PKR expression, thus providing a biological basis for PKLR as a genetic modifier of SCD pain presentation. 3 of the 7 variants are also associated with elevated 2,3-DPG levels in HbSS as well as t50 (a single parameter that depends primarily on sickling kinetics) in HbAS. The variant associations are observed more clearly in HbAS but not in HbSS, possibly because of the much more homogeneous cell population in HbAS. We conclude that PKLR intron variants may directly contribute to the severity and frequency of acute pain episodes in SCD, thus additional studies with larger sample sizes are warranted. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal