Impact of G6PD Α- mutation on hemoglobin function and RBC rheology in sickle cell disease Free

Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency is the most common erythrocyte enzymopathy. Sickle cell disease (SCD) is an autosomal recessive disease affecting approximately 170,000 Americans, with approximately 40% of SCD patients also harboring mutations that decrease G6PD enzyme activity. Despite the common co-inheritance of these mutations, it is unclear how G6PD-deficiency and sickle hemoglobin interact to influence red blood cell (RBC) function. Clinical data has yielded conflicting evidence of the impact of G6PD deficiency on SCD due to low powered studies, lack of clearly defined end points, and failure to control for variables that introduce bias.

SCD causes the RBC intracellular environment to be highly vulnerable to oxidative insults as sickle RBCs produce twice the amount of intracellular hydrogen peroxide (H₂O₂) compared to healthy RBCs. G6PD is responsible for regenerating NADPH, a key substrate for red cell antioxidant enzymes including glutathione peroxidase, which neutralizes H₂O₂. We hypothesize that coinheritance of SCD and G6PD deficiency results in an altered redox environment within the red blood cell, influencing hemoglobin polymerization and RBC rheology, modifying SCD phenotype severity. We have generated a mouse containing the human G6PD A^- mutation with a valine to methionine substitution in the 68^th position that has decreased G6PD activity (10% of WT). This results in increased hemolysis in response to oxidative stress. Crossing this mouse to a SCD Townes mouse allows for the characterization of these co-inherited mutations in the absence of confounding variables. For all experiments, 12–19-week-old male Townes G6PD A^- mice were used. Whole blood was collected via cardiac puncture and in-vitro assays were performed, testing for deformability, point of sickling, and hemoglobin-oxygen affinity.

Townes SS and Townes SS G6PD A^- mice are phenotypically similar at baseline as demonstrated by normal body weight, increased spleen size, and reticulocytosis. Complete blood counts (CBCs) for Townes SS G6PD A^- mice show trends towards increased RBC counts, hemoglobin (Hb), hematocrit (HCT) and red cell distribution width (RDW) compared to Townes SS littermate controls suggesting that decreased G6PD activity affects RBC function. RBCs from Townes SS G6PD A^- mice exhibit trends towards reduced p50 values, defined as the pO₂ at which hemoglobin is 50% saturated, compared to RBCs from Townes SS mice. This suggests a physiologically significant increase in hemoglobin-oxygen affinity which may result in delayed time to hemoglobin polymerization and improved sickling. While Lasser-optical rotational red cell analysis (LORRCA) reveals no difference in baseline deformability between Townes SS and Townes SS G6PD A^- RBCs, and no differences in the maximum elongation index (EI) and points-of-sickling (POS), defined as the pO₂ at 95% of the maximum EI, increased sample size is required.

Together these data indicate that G6PD A^-does not significantly affect baseline measures of red cell health in Townes SS mice. G6PD A^-may paradoxically leave the SCD RBC less vulnerable to deoxygenation by altering parameters that influence RBC rheology like hemoglobin-oxygen affinity yet poised for further damage under oxidative insult. Future work will focus on increasing the power of assays probing RBC parameters, challenging the G6PD deficient phenotype with heme and oxidative stressors, and characterizing metabolomic shifts that may influence RBC rheology in Townes SS G6PD A^- RBCs.